Retrovirus capable of causing AIDS, means and methods for detecting it in vitro

ABSTRACT

The invention relates to a new class of retroviruses, designated by HIV-2, of which samples have been deposited to the ECACC under numbers 87.01.1001 and 87.01.1002 and to the NCIB under numbers 12.398 and 12.399. 
     It relates also to antigens capable to be obtained from this virus, particularly proteins p12, p16, p26 and gp140. These various antigens can be used for the diagnosis of the disease, especially by contacting these antigens with a serum of a patient submitted to the diagnosis. 
     It relates to immunogenic compositions containing more particularly the glycoprotein gp140. Finally it concerns nucleotidic sequences, which can be used especially as hybridization probes, derived from the RNA of HIV-2.

This is a division of application Ser. No. 08/392,613, filed Feb. 22,1995 now U.S. Pat. No. 6,429,306, which is a continuation of applicationSer. No. 08/075,020, filed Jun. 11, 1993 now abandoned, which is acontinuation of application Ser. No. 07/792,524, filed Nov. 18, 1991 nowabandoned, which is a divisional of application Ser. No. 07/462,908,filed Jan. 10, 1990, now U.S. Pat. No. 5,066,782, which is acontinuation of application Ser. No. 07/150,645, filed Nov. 20, 1987,now abandoned, which is a continuation-in-part of application Ser. No.07/003,764, filed Jan. 16, 1987, now U.S. Pat. No. 5,051,496, which is acontinuation-in-part of application Ser. No. 06/933,184, filed Nov. 21,1986, now abandoned, which is a continuation-in-part of application Ser.No. 06/916,080, filed Oct. 6, 1986, now abandoned, and acontinuation-in-part of application Ser. No. 06/835,228, filed Mar. 3,1986, now U.S. Pat. No. 4,839,288, issued Jun. 13, 1989.

The invention relates to a new class of viruses having the capacity tocause lymphadenopathies, which are then capable of being replaced byacquired immune deficiency syndrome (AIDS) in man. The invention alsorelates to antigens capable of being recognized by antibodies induced inman by this new class of virus. It also relates to the antibodiesinduced by antigens obtained from these viruses.

This invention relates, furthermore, to cloned DNA sequences possessingsequence analogy or complementarity with the genomic RNA of theabovementioned virus. It also relates to the methods for preparing thesecloned DNA sequences.

The invention also relates to polypeptides containing amino acidsequences encoded by the cloned DNA sequences.

In addition, the invention relates to applications of these antigens tothe in vitro diagnosis in man of potentials for certain forms of AIDSand, in respect of some of these antigens, to the production ofimmunogenic compositions and vaccinating compositions against thisretrovirus. The invention likewised relates to the applications of theabovementioned antibodies for the same purposes and, for some of them,to their application to the production of acitve principles of drugsagainst these forms of human AIDS.

Finally, the invention relates to the application of the cloned DNAsequences, and of polypeptides obtained from these sequences, as probesin diagnostic kits.

The isolation and characterization of a first retrovirus, known as LAV,whose responsibility in the development of AIDS had been recognized,formed the subject of a description in a paper by F. BARRE-SINOUSSI etal. already in 1983 (Science, vol. 220, No. 45–99, 20, p. 868–871).Application of some extracts of this virus, and more especially of someof its proteins, to the diagnosis of the presence of antibodies againstthe virus was described more especially in European Patent Applicationno. 138.667. Since then, other similar strains and some variants of LAVhave been isolated. Examples which may be recalled are those known bythe names HTLV-III and ARV.

To apply the new rules of nomenclature published in Nature in May 1986,the retroviruses capable of inducing in man the abovementionedlymphadenopathies and AIDS will be given the overall designation “HIV”,an abbreviation of the term “Human Immunodeficiency Virus”. The subgroupof retroviruses formed by LAV and its variants was initially designatedby the terms “LAV type I” or “LAV-I”. The latter subgroup will bedesignated hereinafter HIV-1, it being understood that the term LAV willstill be retained to denote that strain, among the strains of retrovirus(in particular LAAV, IDAV-4 and IDAV-2) belonging to the HIV-1 virusclass which are described in the abovementioned European PatentApplication 138,667, which was used in the comparative experimentsdescribed later, namely LAV_(BRU), which was deposited with theCollection Nationale des Cultures de Micro-organismes (CNCM) (NationalCollection of Micro-organism Cultures) of the Institut Pasteur de Paris,France, on 15th Jul. 1983 under no. I-232.

The new retrovirus which forms the subject of the present patent and thevirus strains which are related to it and which are, like it, capable ofmultiplying in human lymphocytes, formerly known as. “LAV type II” or“LAV-II”, are henceforward known as “HIV-2”, it being understood thatthe designations of certain HIV-2 isolates described later will befollowed by three letters which refer to the patients from whom theywere isolated.

The “HIV-2” group can be defined as a group of viruses having in vitro atropism for human T4 lymphocytes, and which have a cytopathogenic effectwith respect to these lymphocytes when they multiply therein, and theneither cause generalized and persistent polyadenopathies or one of theforms of AIDS. The HIV-2 retroviruses have proved to be different fromthe HIV-1 type viruses under the conditions mentioned later. Like theselatter viruses, they are different from the other human retroviruseswhich are already known (HTLV-I and HTLV-II).

Although there is fairly wide genetic variability in the virus, thedifferent HIV-1 strains isolated to date from American, European,Haitian and African patients have antigenic sites in common conserved ontheir principal proteins, i.e. core protein p25, envelope glycoproteingp110 and transmembrane protein gp41-43. This relationship makes itpossible, for example, for the prototype LAV strain to be used as astrain of antigens for detecting antibodies against all HIV-1 classviruses, in all people who carry them, regardless of their origin. Thisstrain is hence currently used for detecting anti-HIV-1 antibodies inblood donors and patients, in particular by immunofluorescence and inparticular by the technique known as ELISA, “Western Blot” (orimmuno-imprinting) and “RIPA”, an abbreviation forRadioimmunoprecipitation Assay.

However, in a serological study peformed with an HIV-1 lysate onpatients who originated from West Africa, it was observed that some ofthem gave sero-negative or very weakly positive reactions, whereas theyshowed clinical and immunological signs of AIDS.

The cultured lymphocytes of one of these patients were the source of afirst HIV-2 retrovirus isolated, whose structure in electron microscopyand whose protein profile in SDS gel electrophoresis show a resemblanceto those of HIV-1. However, this new retro-virus HIV-2 possesses overallonly a slight relationship to HIV-1, from the standpoint both of theantigenic homology of its proteins and of the homology of its geneticmaterial.

This new retrovirus, or retroviruses having equivalent antigenic andimmunological properties, can hence constitute sources of antigens forthe diagnosis of infection by this virus and the variants which inducean AIDS form of the type which had been observed in the initialinstances in African patients or in people who had spent time in Africa.

Typically, this virus was isolated from the blood, drawn in the presenceof heparin, from a 28-year-old heterosexual patient who had never beentransfused and who was not a drug addict. Since 1983, he had hadsubstantial chronic diarrhoea, and substantial weight loss (17 kg) withintermittent fever. More recently, he had had Candida and Serratiainfections, including an esophageal candidiasis typical of AIDS.

This patient also had anaemia, cutaneous anergy, Lymphopenia and a T4lymphocyte/T8 lymphocyte ratio of 0.15, with a T4 lymphocyte level ofless than 100 per mm³ of serum. His lymphocytes in culture did notrespond to stimulation with phytohaemagglutinin and concanavalin A. Thispatient was also diagnosed as suffering from recurrent bacteriaemia dueto S. enteriditis, cryptosporidioses, infections due to Isospora belliand cerebral toxoplasmoses.

This combination of signs was evidence of “complex symptoms linked withAIDS” or “ARC” (abbreviation for “AIDS-Related Complex”), of the typecaused by HIV-1 virus. These various observations were also inconformity with the criteria applied by the Center of Disease Control(CDC) of Atlanta, USA.

The culturing of the lymphocytes from these patients and the isolationof the retrovirus were performed according to the technique alreadydescribed for the isolation of HIV-1 in the paper by BARRE-SINOUSSI etal. and European Patent Application no. 84/401.834-0.138.667. They arerecalled briefly below. Lymphocytes stimulated for 3 days withphytohaemagglutinin (PHA) were cultured in RPMI 1640 culture medium towhich 10% of foetal calf serum and 10⁻⁵ M β-mecaptoethanol,interleukin-2 and anti-(human interferon α) serum are added.

The production of virus was followed by its reverse transcriptaseactivity. In the culture supernatant, the peak viral activity appearedat between day 14 and day 22, and then decreased. The decline and deathof the cell culture followed. As with HIV-1, sections of lymphocytesinfected with HIV-2 showed, in electron microscopy, virions which hadreached maturity, and viral particles budding at the surface of theinfected cells. The cell lines used for producing the cultures of theseisolated viruses can be, depending on the case, cell lines of the HUT,CEM or MOLT type, or any immortalized lymphocyte Line bearing T4receptors.

The virus was then propagated in cultures of lymphocytes from blooddonors, and then in continuous lines of leukaemic origin, such as HUT78. It was then characterized by its antigens and its nucleic acid asbeing substantially different from HIV-1. The virus was purified asdescribed in the prior documents already mentioned. A first isolate ofthis virus was deposited with the CNCM on 19th Dec. 1985 under no.I-502. It was subsequently designated by the name LAV-II MIR. A secondisolate was deposited with the CNCM on 21st Feb. 1986 under no. I-352.This second isolate has the reference name LAV-II ROD. These isolateswill sometimes be referred to later simply as MIR or ROD.

In a general manner, the invention relates to any variant of the aboveviruses, or any equivalent virus (for example, such as HIV-2-IRMO andHIV-2-EHO, deposited with the CNCM on 19th Dec. 1986 under nos. I-642and I-643, respectively), containing structural proteins which have thesame immunological propeties as those of the HIV-2 viruses depositedwith the CNCM under nos. I-502 or I-532. The definitions of criteria ofequivalence will be returned to later.

The invention also relates to a method for producing the HIV-2 virus orvariants of the latter in permanent cell lines derived from T4lymphocytes, or lymphocytes bearing the T4 phenotype, this methodconsisting in culturing these lines which have been infected beforehandwith HIV-2 virus and, in particular when the level of reversetranscriptase activity has reached a specified threshold, in recoveringthe amounts of virus released into the culture medium.

A preferred permanent line for the purpose of culturing HIV-2 is, forexample, of the HUT 78 cell type. An HUT 78 line infected with HIV-2 wasdeposited on 6th Feb. 1986 with the CNCM under no. I-519. Culturing is,for example, carried out as follows:

The HUT 78 cells (10⁶/ml) are co-cultured with infected normal humanlymphocyte (10⁶/ml). The culture medium is RPMI 1640 with 10% foetalcalf serum. After 15 to 21 days, a cytopathogenic effect is observed inthe HUT 78 cells. The reverse transcriptase is assayed one week afterthis observation, in the culture supernatant. It is then possible tobegin to recover the virus from this supernatant.

Another preferred line for culturing belongs to the lines known underthe designation CEM.

The infection and then the culturing of the infected CEM cells can becarried out, in particular, as follows.

T4 lymphocytes infected beforehand with HIV-2 virus and uninfected cellsof the CEM line are co-cultured for the time required for infection ofthe CEM. The culture conditions are then, moreover, continued in asuitable medium, for example that described below, and when the reversetranscriptase activity of the infected cells has reached a sufficientlevel the virus produced is recovered from the culture medium.

In particular, co-culturing was carried out, under the conditionsspecified below, of human T4 lymphocytes which had been infected fivedays beforehand with a strain of HIV-2 virus originating from a patienthereinafter designated “ROD”, on the one hand, and CEM, on the otherhand.

The infected T4 lymphocytes, activated beforehand withphytohaemagglutinin, proved to possess a reverse transcriptase activityof 5,000 cpm/10⁶ normal T lymphocytes three days after the beginning ofthe infection. Culturing was continued until the measured reversetranscriptase activity reached 100,000 cpm in the supernatant. These T4lymphocytes were then placed in contact with CEM cells (3×10⁶ infectednormal T lymphocytes) and reincubated in the following culture medium:RPMI 1640 containing 2.92 mg/ml of L-glutamine, 10% of decomplementedfoetal calf serum, 2 ug/ml of Polybrene, 0.05% of anti-interferon-alphaserum, 100,000 ug/ml of penicillin, 10 ug/ml of streptomycin and 10,000ug/ml of neomycin.

The culture medium is changed twice weekly.

The measurements of reverse transcriptase activity measured in thesupernatant were as follows:

on day: 0 1.000 (background) on day 15: 20.000 on day 21: 200.000 on day35: 1.000.000

A CEM culture infected with HIV-2 virus was deposited with theCollection Nationale de Cultures de Micro-organismes (CNCM) of theInstitut Pasteur under no. I-537 on 24th Mar. 1986.

A few characteristics of the antigens and nucleic acids involved in thestructure of HIV-2 emerge from the experiments carried out under theconditions described below. They will, in many cases, be better assessedby comparison with the same type of characteristics relating to othertypes of retrovirus, in particular HIV-1 and SIV.

In that which follows, reference will be made to the drawings, in which:

FIGS. 1 a, 1 b and 1 c relate to crossed immuno-precipitationexperiments between sera, respectively, of patients affected with HIV-1and HIV-2, and of rhesus monkeys infected with STLV-III, on the onehand, and viral extracts of HIV-1, on the other hand;

FIGS. 2 a and 2 b show comparative results for the electrophoreticmobilities of the proteins of HIV-1, HIV-2 and STLV-III respectively, inSDS-polyacrylamide gels;

FIG. 3 shows the results of crossed hybridization between genomicsequences of HIV-1, HIV-2 and STLV-III on the one hand, and probescontaining different subgenomic sequences of the HIV-1 virus, on theother hand;

FIG. 4 is a restriction map of the cDNA derived from the RNA of HIV-2ROD;

FIG. 5 is a restriction map of an E2 fragment of the cDNA derived fromHIV-2, this fragment containing a region corresponding to the 3′ LTRregion of HIV-2;

FIG. 6 is a nucleotide sequence of a portion of E2, this sequencecorresponding to the U3/R region of HIV-2;

FIG. 7 shows:

-   -   on the one hand, and schematically, structural elements of HIV-1        (FIG. 7A) and, aligned with a region containing the HIV-1 3′        LTR, the sequence derived from the E2 region of the HIV-2 cDNA,        and    -   on the other hand, the common nucleotides present, respectively,        in the sequence derived from the E2 region of HIV-2 and in the        corresponding sequence of HIV-1, placed in alignment at the cost        of a number of deletions and insertions (FIG. 7B);

FIG. 8 shows schematically the structures of several clones of a phagemodified by several inserts originating from the cDNA derived from HIV-2(clones ROD4, ROD27 and ROD35); sequences derived, in their turn, fromROD4, ROD27 and ROD35, subcloned in a plasmid pUC18, have also beenshown schemetically in this figure, these latter sequences being placedto correspond with the regions of ROD4, ROD27 and ROD35 from which theyrespectively originate;

FIG. 9 shows the relative intensities of hybridization between

a/ eleven fragments removed from different regions of the complete HIV-1genome (the fragments being shown schematically at the bottom of thefigure), on the one hand, and the HIV-2 cDNA, present in ROD4, on theother hand, and

b/ fragments originating from HIV-2 with the same cDNA.

Generelly, the HIV-2 antigens used in the comparative tests, thedescription of which follows, originate from the HIV-2 MIR straindeposited with the CNCM under no. I-502, and the DNA sequences derivedfrom the genomic DNA of HIV-2 originate from the strain HIV-2 RODdeposited with the CNCM under no. I-352.

I—Antigens, in Particular Proteins and Glycoproteins

The virus initially cultured in HUT 78 was labelled metabolically with[³⁵S]systeine and [³⁵S]methionine, the infected cells being incubated inthe presence of these radioactive amino acids in culture medium devoidof the corresponding unlabelled amino acid, for a period of 14 to 16hours, especially according to the technique described in the paperdesignated as reference (21) in the bibliography presented at the end ofthe description, as regards the labelling with [³⁵S]cysteine. Thesupernatant is then clarified and the virus then ultracentrifuged forone hour at 100,000 g on a cushion of 20% sucrose. The principalantigens of the virus separated by electrophoresis in a polyacrylamidegel (12.5%) under denaturing conditions (SDS), or in a gel composed ofpoly-acrylamide (10%)+bisacrylamide (0.13%) with SDS (0.1% finalconcentration). The following coloured markers are used as molecularweight references:

myosin: 200 kd phosphorylase B: 97.4 kd BSA: 68 kd ovalbumin: 43 kdα-chymotrypsin: 25.7 kd β-lactoglobulin: 18.4 kd lysozyme: 14.3 kd

Other molecular weight markers were used in other experiments. Thisapplies, in particular, to FIGS. 1 a, 1 b and 1 c, which refer to otherknown molecular weight markers (under the letter M in these figures).The antigens are still more readily distinguished afterimmuno-precipitation (RIPA) or by immuno-imprinting (Wastern blot),using the antibodies present in the patient's serum their apparentmolecular weights, determined by their apparent migrations, are veryclose to those of the HIV-1 antigens.

It is generally specified that, in the text which follows, the numberswhich follow the designations “p” and/or “gp” correspond to theapproximate molecular weights of the corresponding proteins and/orglycoproteins, divided by 1000. For example, p36 has a molecular weightof the order of 36,000. It is, however, understood that these molecularweight values can vary within a range which can reach 5%, 10% or evenmore, depending on the techniques used for the determination of thesemolecular weights.

Repetition of the experiments enabled the apparent molecular weights ofthe HIV-2 antigens to be determined more accurately. Thus, it was foundthat the molecular weights of the three core proteins, which hadinitially been assigned molecular weights of the order of 13,000, 18,000and 25,000, respectively, in fact had apparent molecular weights closerto the following values: 12,000, 16,000 and 26,000, respectively. Theseproteins are hereinafter designated by the abbreviations p12, p16 andp26.

The same considerations apply to the existence of protein orglycoprotein bands whose apparent molecular weights were assessed atvalues which could range from 32,000 to 42,000–45,000. Repetition of themeasurements finally enabled a band corresponding to an apparentmolecular weight of 36,000 to be precisely defined. In the text whichfollows, this band is designated by the abbreviation p36. Another bandat 42,000–45,000 (p42) is consistently observed also. One or other ofp36 or p42 probably constitutes a transmembrane glycoprotein of thevirus.

A major envelope glycoprotein having a molecular weight of the order of130–140 kd is consistently observed this glycoprotein is designatedhereinafter by the term gp140.

It is appropriate to note that, in general, the molecular weights areassessed with an accuracy of ±5%, this accuracy even being capable ofbecoming a little lower for antigens of high molecular weight, as wasfournd for gp140 (molecular weight of 140±10%). This group of antigens(when they are labelled with [³⁵ S]cysteine is only faintly recognized,if at all, by sera of patients containing anti-HIV-1 antibodies in thedetection systems used in the laboratory or by the use of testsemploying HIV-1 lysates, such as those marketed by DIAGNOSTICS PASTEURunder the name “ELAVIA”. Only the p26 protein was weaklyimmunoprecipitated by such sera. The envelope protein was notprecipitated. The serum of the patient infected with the new virus(HIV-2) faintly recognizes a p34 protein of HIV-1. In the detectionsystem used, the other HIV-1 proteins were not recognized.

In contrast, HIV-2 possesses some proteins which show some immunologicalrelationship with comparable structural proteins or glycoproteins,separated under similar conditions from a retrovirus recently isolatedfrom captive macaques of the rhesus species, whereas this immunologicalrelationship tends to become obliterated for other proteins orglycoproteins. This latter retrovirus, which is presumed to be theetiological agent of AIDS in monkeys, was designated by theinvestigators who isolated it [bibliographic references (16–18) below]by the name “STLV-III_(mac)”. For convenience of reference, it will bedesignated in the text which follows simply by the term “STLV-III” (oralternatively by the term SIV, an abbreviation for “SimianImmunodeficiency Virus”).

Another retrovirus, designated “STLV-III_(AGM)” or SIV_(AGM)), has beenisolated in wild green monkeys. However, in contrast to the viruspresent in rhesus monkeys, the presence of “STLV-III_(AGM)” does notappear to induce an AIDS-type disease in African green monkeys.

Nevertheless, the immunological relationship of the structural proteinsand glycoproteins of HIV-2 on the one hand and the STLV-III_(mac) andSTLV-III_(AGM) retro-viruses on the other hand, and consequently therelationship of their nucleic acid sequences, remains limited.Experiments have enabled a first distinction to be established betweenthe retroviruses capable of infecting man or monkeys; the followingemerges;

The HIV-2 virus does not multiply in chronic fashion in the lymphocytesof rhesus monkeys when it has been injected in vivo and under workingconditions which permit the development of the STLV-III_(mac) virus, ashave been described by N. L. Letvin et al., Science (1985), vol. 230,71–75.

This apparent inability of HIV-2 to develop in monkeys under naturalconditions enables the HIV-2 virus, on the one hand, and the STLV-IIIvirus isolates, on the other hand, to be differentiated biologically.

Employing the same techniques as those recorded above, it was found thatit was also possible to obtain the following from STLV-III:

a principal p27 core protein, having a molecular weight of the order of27 kilodaltons,

a major envelope glycoprotein, gp140,

a p32 protein, probably transmembrane, which is not observed in RIPAwhen the virus has been labelled beforehand with [³⁵]cysteine, but whichcan be observed in immuno-imprinting experiments (Western blots) in theform of broad bands.

The major envelope glycoprotein of HIV-2 has proved to beimmunologically closer to the major envelope glycoprotein of STLV-IIIthan to the major envelope glyco-protein of HIV-1.

These findings apply not only in respect of the molecular weights,130–140 kilodaltons for the major glycoproteins of HIV-2 and STLV-IIIcompared with approximately 110 for the major envelope glycoprotein ofHIV-1, but also in respect of the immunological properties, since seradrawn from patients infected with HIV-2, and more especially antibodiesformed against the HIV-2 gp140, recognize the STLV-III gp140 whereas, incomparable experiments, the same sera and the same antibodies to HIV-2do not recognize the HIV-1 gp110. However, anti-hiv-2 do not recognizethe HIV-1 gp110. However, anti-HIV-1 sera which have never reacted withthe HIV-2 gp140s precipitate a [³⁵S]cysteine-labelled 26 kd proteinpresent in extracts of HIV-2.

The major core protein of HIV-2 appears to have an average molecularweight (approximately 26.000) intermediate between that of the HIV-1 p25and the p27 of STLV-III.

These observations are derived from experiments carried out with viralextracts obtained from HIV-2 isolated from one of the above-mentionedpatients. Similar results have been obtained with viral extracts ofHIV-2 isolated from the second patient.

Cells infected, respectively, with HIV-1, HIV-2 and STLV-III wereincubated in a medium containing 200 μCi/ml of [³⁵S]cysteine in a mediumfree from unlabelled cysteine for 16 hours. The clarified supernatantswere centrifuged at 60,000 g for 90 minutes. The pellets were lysed inan RIPA buffer (1), immunoprecipitated with different sera and thensubjected to electrophoresis on polyacrylamide gel charged with sodiumdodecyl sulphate (SDS-PAGE).

The results observed are illustrated by FIGS. 1 a, 1 b and 1 c.

FIG. 1 a shows the observed results of immuno-precipitation between aviral extract of HIV-1 obtained from a CEM C1.13 cell line and thefollowing sera, respectively:

anti-HIV-1-positive serum (band 1),

serum obtained from the first patient mentioned above (band 2),

serum of a healthy African carrier of anti-HIV-1 antibodies (band 3),

serum obtained from a macaque infected with

STLV-III (band 4), and

serum of the second patient mentioned above (band 5).

In FIG. 1 b, there are recorded the observed results ofimmunoprecipitation between the HIV-2 antigens obtained from the firstpatient, after prior culture with HUT-78 cells, and different sera, moreespecially the serum of the abovementioned first patient (band 1), theanti-HIV-1-positive serum (band 2), the serum of the macaque infectedwith STLV-III (band 3) and the serum of the abovementioned secondpatient (band 4).

Finally, FIG. 1 c illustrates the observed results ofimmunoprecipitation between the antigens of an STLV-III isolate obtainedfrom a macaque having a simian AIDS. The sera used, to which the bands 1to 5 refer, are the same as those recorded above in relation to FIG. 1a.

M refers to the markers myosin (200 kd), galactosidase (130 kd), bovineserum albumin (69 kd), phosphorylase B (93 kd), ovalbumin (46 kd) andcarbonic anhydrase (30 kd).

FIGS. 2 a and 2 b show comparative results for the electrophoreticmobilities of the proteins of HIV-1, HIV-2 and STLV-III.

FIG. 2 a relates to the experiments carried out with extracts of viruslabelled with [³⁵ S]ψysteine, after immunoprecipitation on SDS-PAGE. Thedifferent bands relate to the following virus extracts: virus obtainedfrom patient 1 and immunoprecipitated by the serum originating from thesame patient (band 1), extract of the same virus immunoprecipitated witha negative control serum originating from a person not carryinganti-HIV-1 or anti-HIV-2 antibodies (band2), extract of STLV-III virusimmunoprecipitated with a serum originating from a macaque infected withSTLV-III (band 3), immunoprecipitation observed between extracts of thesame virus and a negative control serum (band 4), and extract of HIV-1immunoprecipitated with the serum of a European patient infected withAIDS.

FIG. 1 b shows the results obtained in Western blot (immuno-imprinting)experiments. Cell lysates originating from uninfected or infected HUT-78cells were subjected to electrophoresis on SDS-PAGE, and thentransferred electrophoretically to a nitrocellulose filter before beingreacted with the serum of the above-mentioned first patient (serumdiluted 1/100). The nitrocellulose filter was then washed and thedetection of the bound antibodies visualized with ¹²⁵I-labelled goatanti-human IgG.

The spots observed in bands 1, 2 and 3 relate, respectively, to theagglutination experiments between the abovementioned serum and extractsof uninfected HUT-78 cells (band 1), extracts of HUT-78 cells infectedwith an HIV-2 virus (band 2) and extracts of HUT-78 cells infected withSTLV-III (band 3). The numbers which appear in the margins beside eachof the bands correspond to the approximate molecular weights of the mostrepresentative viral proteins (molecular weights in kilodaltons).

II—Nucleic Acids

-   -   1/ The RNAs of the HIV-2 retrovirus

The RNA of the virus, deposited on a filter according to the “spot blot”technique, did not hybridize, under stringent conditions, with the DNAof HIV-1.

By “stringent conditions”, there are understood the conditions underwhich the hybridization reaction between the RNA of the HIV-2 and thechosen probe, radio-actively labelled with ³²P (or labelled in adifferent manner), followed by the washing of the probe, are carriedout. The hybridization, on a membrane, is carried out at 42° C. in thepresence of an aqueous solution particularly of 50% formamide(volume/volume) in 0.1% SDS/5×SSC for 18 hours. The membrane on whichthe hybridization reaction has been carried out is then washed at 65° C.in a buffer containing 0.15% of SDS and 0.1×SSC.

By “non-stringent conditions”, there are understood the conditions underwhich the hybridization reaction and the washing are carried out. Thehybridization is carried out by bringing into contact with the chosenprobe, labelled with ³²P (or otherwise labelled), namely at 42° C. in a5×SSC buffer, 0.1% SDS, containing 30% of formamide for 18 hours. Thewashing of the membrane is carried out at 50° C. with a buffercontaining 0.1“ ” of SDS and 2×SSC.

Hybridization experiments were also carried out with a hybridizationprobe consisting of a recombinant plasmid pBT1 obtained by cloning theDNA of HIV-1 originating from λJ19 (Cell 1985, vol. 40, p. 9) in thevector pUC18. Under non-stringent conditions, only very weakhybridization was observed between the RNA of HIV-2 and the cloned DNAderived from HIV-1.

Other probes containing cloned sequences of HIV-1 were used:

a/ single-stranded probes of subgenomic DNA of HIV-1, produced fromsubclones of the HIV-1 genome and inserted in phage M13. The clonedregions related to the protease gene or the “endonuclease” gene.

Only one probe of the endonuclease region of HIV-1 (nucleotide sequencebetween bases nos. 3760 and 4130) gave a weak hybridization undernon-stringent conditions with HIV-2. The “protease” probe (HIV-1nucleotide sequence between bases nos. 1680 and 1804) did not hybridizeeven under non-stringent conditions with HIV-2.

b/ A probe pRS3, consisting of the sequence coding for the “envelope”region of HIV-1 (subcloning in pUC18) did not give hybridization undernon-stringent conditions with HIV-2.

The “spot blot” technique is also known as “dot blot” (transfer byspots).

Further results of hybridization between genomic RNAs of HIV-1, HIV-2and STLV-III, on the one hand, and probes containing differentsubgenomic sequences of the HIV-1 virus, on the other hand, appear inFIG. 3.

The supernatants of the different culture media (in the proportion of0.5 to 1 ml for each spot) were centrifuged for 5 minutes at 45,000revolutions per minute; the pellets were resuspended in an NTE buffercontaining 0.1% of SDS and deposited on a nitrocellulose filter. Thelatter was pre-soaked in a 2×SSC medium (0.3 M NaCl, 0.03 M sodiumcitrate). After baking (for 2 hours at 80° C.), the filters werehybridized with various probes contraining genomic sub-fragments ofHIV-1, under non-stringent conditions (30% formamide, 5×SSC, 40° C.),washed at 50° C. with a 2×SSC solution containing 0.1% of SDS and thenautoradiographed for 48 hours at −70° C. with enhancing screens.

The probes 1–4 are single-stranded probes obtained by the “prime cut”method as described in (25). Briefly, the single-stranded fragmentsoriginating from the M13 virus and bearing subgenomic HIV-1 inserts (30)were ligated to oligomeric fragments (17 nucleotides) originating fromM13 (BIOLABS). The complementary strand was then synthesized with Klenowenzyme in a TM buffer (10 mM Tris, pH 7.5, 10 mMMgCl₂) in the presenceof dATP, dGTP, dTTP and dCTB, labelled with ³²P at the alpha-position(Amersham, 3000 Ci/mmol). The DNA was then digested with the appropriaterestriction enzymes, heat denatured and subjected to electrophoresis ona denaturing polyacrylamide gel (containing 6% of acrylamide, 8 M ureain a TDE buffer). The gel was then autoradiographed for 5 minutes. Theprobe was then cut out and eluted in a 300 mM NaCl, 0.1% SDS buffer.Specific activities (SA) of these single-stranded probes were estimatedat 5×10⁸–10⁹ disintegrations per minute/microgram (dpm/ug).

The characteristic sequences present in the different probes were asfollows:

Probe 1: nucleotides 990–1070, Probe 2: nucleotides 980–1260, Probe 3:nucleotides 2170–2240 Probe 4: nucleotides 3370–3640.

The numbering of the above nucleotides are those envisaged in the paperunder reference (30).

Lastly, the probe 5 consists of a plasmid pUC18 bearing the EcoR1-Sac1fragment of the HIV clone in λJ19 (31), which was subjected to nicktranslation to obtain an SA of approximately 10⁸ dpm/μg.

The relative arrangements of the subgenomic fragments present in theprobes with respect to the whole HIV-1 genome are shown schematically inFIG. 3. The different spots correspond, respectively, as follows:

spot A: a virus is obtained from a culture of CEM C1.13 cells infectedwith HIV-1,

spot B: a virus is obtained from HUT-78 cells infected with STLV-III,

spots C and D: isolates obtained, respectively, from the viruses of theabovementioned two African patients,

spot E: negative control cell extract obtained from uninfected HUT-78cells,

spot F: virus obtained from a patient from Zaīre suffering from AIDS,which had been cultured in normal T lymphocytes in the presence of TCGF.

All the spots were obtained with an amount of virus corresponding to25,000 dpm of reverse transcriptase activity, except for the spots C:15,000 dpm.

The following observations were made:

The genomic RNAs of the two HIV-2 isolates obtained from purified viralparticles did not hybridize with any of the probes under the stringentconditions described above, although the viral particles were isolatedand purified from culture supernatants of highly infected cells showingevidence of high reverse transcriptase activity.

Under the non-stringent conditions described above, the followingobservations were made: all the probes hybridized intensely with thegenomic RNAs obtained from the control HIV-1 preparations and fromanother isolate obtained from a patient from Zaīre suffering from AIDS.

Two of the probes obtained (nucleotides 990–1070 and 990–1260, bothoriginating from the gag region of HIV-1) hybridized slightly with thespots from extracts of the HIV-2 retroviruses; only one of these twoprobes (nucleotides 990–1260) also showed slight hybridization with theSTLV-III spot (FIG. 3). As regards the probe containing a fragment ofthe pol region (nucleotides 2170–2240), hybridization was observed withSTLV-III and, albeit much more weakly, with the RNA of HIV-2. The otherprobe of the pol region (nucleotides 3370–3640) did not givehybridization with any of the HIV-2 and STLV-III spots.

Lastly, the probe modified by nick translation and containing the entireenv gene and the LTR (nucleotides 5290–9130) of HIV-2 did not hybridizeeither with the RNAs of STLV-III or with those of HIV-2.

It will also be noted that anoter probe which contained the 5′ end ofthe Pol reading frame of HIV-1 (corresponding to the protease region)did not hybridize either with the RNAs of HIV-2 or with the RNAs ofSTLV-III.

It consequently also results from the foregoing that the HIV-2 virusappears more remote, from the structural standpoint, from the HIV-1virus than it is from STLV-III. HIV-2 nevertheless differs significantlyfrom STLV-III, which bears out the different results observed in respectof the infective capacities of the HIV-2 viruses, which are virtuallynil in monkeys, compared with the unquestionable ineffective capacitiesof STLV-III viruses in these same specites of monkeys.

The restriction maps and the genomic RNA sequences of HIV-2, or of thecDNAs obtained from these genomic RNAs are accessible to those versed inthe art, since the strains of HIV-2 deposited with the CNCM can, aftersuitable multiplication, provide him with the genetic equipment requiredfor the determination of these restriction maps and nucleotidesequences. The conditions under which the restriction map of the genomeof one of the HIV-2 isolates of this invention were established, and theconditions under which certain portions of cDNA derived from thesegenomes were sequenced, are described below.

The restriction map of the genome of a retro-virus which isrepresentative of HIV-2 retroviruses is shown in FIG. 4. The restrictionmap of a substantial fragment of this cDNA is shown in FIG. 5. Finally,a portion of this latter fragment has been sequenced.

This sequence, and a number of the restriction sites which it contains,are shown in FIG. 6. The cloned whole cDNA—or cloned fragments of thiscDNA—can themselves be used as specific hybridization probes.

-   -   2/ cDNA and fragments of this cDNA derives, respectively, from        the RNA of HIV-2

The conditions under which the abovementioned cDNA was obtained aredescribed below.

The first stage of manufacture of this cDNA comprised the production ofan oligo(dT) serving as a primer or of an initiator cDNA strand, bycarrying out an endogenous reaction activated by a detergent, using thereverse transcriptase of HIV-2, on purified virions obtained fromsupernatants of infected CEM cells. The CEM cell line was alymphoblastoid CD4+ cell line described by G. E. Foley et al. in Cancer18: 522–529 (1965), which is considered to be incorporated herein byreference. These CEM cells used are infected with an ROD isolate, whichwas shown to produce substantial amounts of HIV-2 continuously.

After the synthesis of the second strand (in the presence of nucleotidesand a bacterial DNA polymerase), the double-stranded cDNAs were insertedinto a bacterial phage vector M13 TG130. A phage library of 10⁴recombinant M13 phages was obtained and subjected to an in situscreening with an HIV-1 probe. The latter contains a 1.5 kb fragmentoriginating from the 3′ end of the cDNA derived from the RNA of the LAVisolate (shown in FIG. 7A). Approximately 50 positive plaques weredetected, purified and characterized by crossed hybridization of theinserts and sequencing of the ends.

This procedure enabled different clones to be isolated, containingsequences approximately complementary to the 3′ end of thepolyadenylated RNA of the LTR [abbreviation for “long terminal repeat”of HIV-1, described by S. Wain Hobson et al. in Cell 40: 9–17 (1985)]region, considered to be incorporated herein by reference.

The largest of the inserts of the group of M13 clones in question, whichhybridize with the 3′ LTR region of HIV-1, as an approximately 2 kbclone designated E2. Like the 3′ LTR region of HIV-1, the clone E2contains an AATAAA signal situated approximately 20 nucleotides upstreamfrom a poly(A) terminal portion, and a 3′ LTR region corresponding tothat of HIV-2. After partial sequencing, this 3′ LTR region of HIV-2proved to possess a distant relationship with the homologous region ofHIV-1.

FIG. 5 is a restriction map of the fragment of E2 (elongated rectangulararea) incorporated in plasmid pSPE2 which contains it. It comprises partof the R region and the U3 region of HIV-2. The drawing does not showthe boundaries of the R and U3 regions.

The sequence of part of E2 is shown in FIG. 6. The positions of specificrestriction sites are indicated therein. The small degree ofrelationship between the 3′ LTR regions of HIV-1 and HIV-2 isillustrated in FIG. 7. In effect, only approximately 50% of thenucleotides of the two LTR sequences can be placed in alignment(approximately 50% sequence homology), at the cost of some insertions ordeletions. In comparison, the sequence homology of the correspondingregions the different variant American and African isolates of HIV-1 isgreater than 95%, without insertion or deletion.

The clone E2 was used as a specific probe for HIV-2, for theidentification on a hybridization filter of the sequences originatingfrom HIV-2 and present in other clones.

This probe also detects the genomic RNA of HIV-2 under stringentconditions. It likewise permits detection, by the so-called “Southernblot” method on the DNA of CEM or similar cells infected with an RODisolate or with other HIV-2 isolates. No signal is detected under thesame stringency conditions in tests of hybridization of this probe withcDNAs originating from uninfected cells or from cells infected withHIV-1. These results confirmed the exogenous nature of HIV-2 withrespect to HIV-1. An approximately 10 kb species, probably correspondingto the non-integrated viral DNA, was detected as a principal constituentin the undigested DNA of cells infected with HIV-2. Anoter DNA having anapparent size of 6 kb, possibly corresponding to a circular form of theviral DNA, was also detected.

The other portions of the HIV-2 genome were also identified. For thispurpose, a genome library was constructed in phage lambda L47. Phagelambda L47.1 has been described by W. A. M. Loenen et al. in Gene 10249–259 (1980), which publication is considered to be incorporatedherein by reference.

The genome library is constructed with fragments obtained by digestin ofthe DNA originating from the CEM cell line infected with HIV-2 ROD,after digestion with the enzyme Sau3AI.

Approximately 2×10⁶ recombinant plaques were screened in situ with aclone containing the labelled E2 insert of the HIV-2 cDNA. Tenrecombinant phages were detected on plaques and purified. Therestriction maps of three of these phages, characterized by theircapacity for “Southern blot” hybridization with the E2 insert understringent conditions, as well as with subgenomic probes of HIV-1 undernon-stringent conditions.

A clone bearing a 9.5 kb insert and derived from the whole circularviral DNA, containing the complete HIV-2 genome, was identified. It wasdesignated “Lambda ROD 4”. The other two clones, Lambda ROD 27 andLambda ROD 35, derived from integrated proviruses, bear LTR sequences ofthe viral coding sequences and adjacent cell DNA sequences. Thedifferent sequences are shown in FIG. 8.

Fragments of the Lambda clones were subcloned in plasmid vector pUC18.The fragments originating from λ ROD 4, λ ROD 27 and λ ROD 35, andsubclones respectively, in the abovementioned plasmid vector, are alsoseen in FIG. 8. The following subclones were obtained:

pROD 27–5, derived from Lambda ROD 27, contains a 5.2 kb region of theHIV-2 genome and adjacent cell sequences (5′ LTR and 5′ coding viralsequence around an EcoRI site);

pROD 4.8, derived from Lambda ROD 4, contains an approximately 5 kbHindIII fragment. This fragment corresponds to the central portion ofthe HIV-2 genome;

pROD 27-5′ and pROD 4.8 contain HIV-2 inserts chich overlap each other;

pROD 4.7 contains a 1.8 kb HindIII fragment of Lambda ROD 4; thisfragment is placed in the 3 direction with respect to the subclonedfragment in pROD 4.8, and contains approximately 0.8 kb of coding viralsequences and a portion situated between the BamHI and HindIII cloningsites of the left arm of phage Lambda (Lambda L 47.1);

pROD 35 contains all the HIV-2 coding sequences in the 3 direction withrespect to the EcoRI site, the 3′ LTR end and approximately 4 kb ofadjacent nucleotide sequences of cellular origin:

pROD 27-5′ and pROD 35, present in E. coli HB 101, were deposited onNov. 21, 1986 with the CNCM under 1-626 and I-633;

pROD 4.7 and pROD 8, present in E. coli TG1, were deposited on Nov. 21,1986 with the CNCM, respectively, under nos. I-627 and I-628.

The complete HIV-2 ROD genome, the restriction map of which is seen inFIG. 4, was reconstituted from pROD 35, linearized beforehand withEcoRI, and pROD 27-5′. The EcoRI insert of pROD27-5 was ligated in thecorrect orientation in the EcoRI site of pROD 35.

The degree of relationship between HIV-2 and the other human or simianretroviruses was assessed by mutual hybridization experiments. Therelative homology between the different regions of HIV-1 and HIV-2genomes was determined by tests of hybridization of fragmentsoriginating, respectively, from cloned HIV-1 genome and fromradioactively labelled lambda ROD 4. The relative positions of thesefragments (numbered from 1 to 11) with respect to the HIV-1 genome areseen at the bottom of FIG. 9.

Even under very low stringency conditions (Tm-42° C.), the HIV-1 andHIV-2 genomes hybridize only at the level of their respective gag genes(spots 1 and 2), reverse transcriptase regions in pol (spot 3), endregions of pol, Q (or sor) genes (spot 5) and F (or 3′ orf) genes and 3′LTR (spot 11). The HIV-1 fragment used for detecting the first cDNAclones of HIV-2 corresponds to the subclone of spot 11, which hybridizesrelatively well with HIV-2 under non-stringent conditions. A signaloriginating from spot 5 is the only one which persists after stringentwashing. The envelope gene, the tat gene region and part of pol appearto be highly divergent. These data, as well as the sequence obtainedwith LTR (FIG. 3), demonstrate that HIV-2 is not (at all events, asregards its envelope) a variant of HIV-1.

It is observed that HIV-2 is more closely related to SIV [described byM. D. Daniel et al in Science 228 1201–1204 (1985)], which must beconsidered to be incorporated herein by reference] than it is to HIV-1.

All the proteins of SIV, including the envelope protein, areimmunoprecipitated by sera of patients infected with HIV-2, while theserological cross-reactivity of HIV-1 and HIV-2 is limited to the coreproteins. However, SIV and HIV-2 can be distinguished by the differencesmentioned above in respect of the molecular weights of their proteins.

As regards the nucleotide sequences, it is also noted that HIV-2 isrelated to SIV.

Furthermore, the characterization of HIV-2 also makes it possible todemarcate the region of the envelope glycoprotein which is responsiblefor the binding of the virus to the surface of the target cells and thesubsequent internalization of the virus. The interaction takes place viathe CD4 molecule itself, and it appears that HIV-1 and HIV-2 use thesame receptor.

Thus, although there are large differences between the env genes ofHIV-1 and 2, the restricted homologous regions of the envelopes of thetwo forms of HIV can be considered to be constituents of binding to acommon receptor of T4 lyphocytes. These sites are called on to formepitopes bearing the immunogenicity of peptides which might be used toelicit in man a protective immunoresponse against HIV viruses.

Advantageous sequences for forming probes in hybridization reactionswith the genetic material of patients carrying viruses or proviruses, inparticular for detecting the presence of HIV-2 virus RNA in theirlymphocytes, contain a nucleotide sequence resulting from thecombination of 5 kb HindIII fragments of ROD 4 and E2 cDNA. Theexperiments can be carried out by all methods, in particular by the“Northern blot”, “Southern blot” and “dot blot” techniques.

Further characteristics of the invention will also emerge, withoutimplied limitation, in the course of the description which follows ofexamples of identification of certain portions of the retroviral genomeand of the production of a number of recombinant DNAs involving variousportions of a cDNA derived from the retroviral genome of HIV-2.

EXAMPLES Example I

DNA Probe, for Use in Kits for Diagnosis of HIV-2

A cDNA complementary to the genome RNA, obtained from purified virions,was prepared by the following method:

The supernatant obtained after 48 hours' culturing of CEM cells infectedwith an HIV-2 ROD isolate of HIV-2 was ultracentrifuged. Thecentrifugation pellet containing the virion was centrifuged on a sucrosegradient to form a new centrifugation pellet, substantially by the samemethod as that described in European Patent Application84/401.234-0.138.667, already mentioned.

The purified HIV-2 preparation was used for the synthesis of cDNA,employing an endogenous reaction activated by a detergent.

In summary, the virion preparation was added to a reaction mixturecontaining 50 mM Tris-HCL, 5 mM MgCl₂, 10 mM DTT, 0.025% of thedetergent marketed under the name TRITON, and 50 μM of each of the 4deoxynucleoside triphosphates and an oligo(dT) initiator. The reactionwas carried out for 90 minutes at 37° C.

After extraction with phenol of the proteins present in the firstreaction medium, the second cDNA chain was synthesized in the presenceof RNAse, E. coli DNA polymerase 1 and the 4 deoxynucleotides, for 1hour at 15° C. and 1 hour at 22° C. Blunt ends were created on thisdouble-stranded cDNA by the action of T4 DNA polymerase. All thereagents for this procedure are commercially available (AMERSHAM cDNAkit) and were used as recommended by the supplier.

After (1) ligation of adapters (linkers) containing an EcoRI site(marketed by Pharmacia) to the blunt ends of the cDNA in the presence ofa T4 DNA ligase (marketed by BIOLABS), (2), digestion of these linkerswith the restriction endonuclease EcoRI, and (3) removal of the linkerfragments by gel filtration (gel column marketed under the nameULTROGEL) on AcA 34 (LKB-IBF), the cDNA is inserted in an M 13 TG 130vector cleaved with EcoRI. A library of cDNAs was obtained aftertransformation of E. coli strain TG1. Approximately 10⁴ recombinant M13plaques were obtained.

To select, in the cDNA library, recombinant M13 clones containing theHIV-2 cDNA, the technique of plaque hybridization was used. The DNA ofthe M13 plaques was transferred to nitrocellulose filters and hybridizedwith subgenomic HIV-1 probes derived from the “lambda J19” clone of anLAV (or HIV) virus decribed in the European patent application. Thisprobe contained an insert consisting of a portion having an approximatelength of 1500 base pairs (bp) of HIV-1 DNA. This insert was bounded bytwo HindIII restriction sites, respectively, inside the open readingframe of the “env” gene and in the R segment of the 3′ LTR end of HIV-1.This probe contained the 3′ end of the env gene, the whole F gene, theU3 segment and a portion of the R segment of the LTR, having anapproximate length of 1500 base pairs (bp).

The probe containing the 1.5 kg HindIII insert was labelled with[³²P]-dCTP and -dTTP (3000 Ci×10⁻³ mole) by incubating the probe in thepresence of initiators and Klenow DNA polymerase I for 4 hours at 15° C.(using an AMERSHAM kit). The tests of hybridization of the cDNA clonesof the library were performed overnight under low stringency conditions,in a solution of a hybridization medium containing 5×SSC, 5× Denhart,25% of formamide, 100 μg/ml of denatured salmon sperm DNA and thelabelled probe (2×10⁷ cpm with a specificity of 10⁹ cpm/μg) at 37° C.The filters were subjected to three washing stages, successively in thepresence of the three solutions whose compositions are stated asfollows:

Washing no. 1: 5×SSC, 0.1% SDS, at 25° C. for 4×15 minutes

Washing no. 2: 2×SSC, 0.1% SDS, at 42° C. for 2×30 minutes

Washing no. 3:0.1×SSC, 0.1% SDS, at 65° C. for 2×30 minutes Each washingis followed by autoradiography of the filters.

Several positive clones were detected after ashing no. 1 and were stilldetected after washing no. 2. However, all the signals disappeared afterwashing no. 3. This indicates that the positive clones had only a weakrelationship with the HIV-1 genome, which was nevertheless sufficient toperform the abovementioned selection. The positive clones weresubcultured, redeposited on plaques and again hybridized with the sameprobe under the stringency conditions corresponding to washing no. 1.Most of them were still positive.

The clones were also selected using a total human DNA probe underconditions of moderate stringency and by hybridization in 5×SSC, 5×Denhart and 40% formamide, followed by washing in 1×SSC, 0.1% SDS at 50°C. None of the previously positive clones was detected, and consequentlydid not correspond to specific repeated DNA or to the cDNA of theribosomal RNA.

The positive M13 recombinant clones were cultured in a liquid medium andcharacterized as follows:

(1) Size of their Insertion:

An M13 single-stranded type DNA was obtained from each individual clone,and the synthesis of the second strand was performed with an M13 17-merinitiator sequence and the Klenow enzyme. The inserts were excised usingEcoRI (BOEHRINGER) and analysed by agarose gel electrophoresis. Themajority of the inserts contained from 200 to 600 and 200 bp, with theexception of the clone designated E2.1, which had an approximate lengthof 2 kbp.

(2) Analysis of the Nucleotide Sequence

Several clones were partially sequenced using the dideoxy method ofSanger et al., described in Proc. Natl. Acad. Sci. 74:5463–7 (1977),which forms part of the present description. Various independent clonescontained similar nucleotide sequences, with the exception of thepoly(A) chains at their 3′ ends, the lengths of which were different.These results demonstrate that these cDNA clones were derived from theRNA template. Detailed sequence analysis of these cDNA clones, includingthe 3′ end of the HIV-2 genome, showed a limited relationship withHIV-1.

(3) Hybridization with the Genomic RNA and DNA of HIV-2:

(a) Production of the Genomic RNA of HIV-2

An infected supernatant was centrifuged (50,000 revolutions, 30minutes). The pellet of the deposit was resuspended in 10 mM Tris pH7.5, 1 mM EDTA, 0.1% SDS. One of the insertion clones, F1.1, waslabelled and used as a probe for hybridization with the genomic RNA ofdifferent viral isolates, according to the “dot-blot” technique.

The “dot-blot” technique comprised the following stages:

(i) Depositing the sample (HIV-2 lysate) in spots on a nitrocellulosemembrane soaked beforehand in 20×SSC (3 M NaCl, 0.3 M sodium citrate)and dried in the air, (ii) baking the membrane for 2 hours at 80 C, and(iii) performing the hybridization.

This hybridization was performed under high stringency conditions(5×SSC, 5× Denhart, 50′ formamide at 42° C.). It was followed by washingin 0.1×SSC, 0.1% SDS at 65° C. Under these conditions, the probehybridizes strongly to the spots originating from two independentisolates of HIV-2, including LAV-II ROD, from which the cloned cDNAoriginated. A weak hybridization signal was detected with the spotformed by STLV-III mac [Simian T-lymphotropic Virus (also known as“SIV”), type III macaque], and no hybridization was detected with theHIV-1 isolates.

The “Southern blot” experiments, employing the clone E2.1 containing the2 kb insert as a ³²P-labelled probe, did not reveal any hybridizationwith the DNAs of uninfected cells, but detected bands in detached cellsinfected with HIV-2, under high stringency conditions. HIV-2 showspolymorphism at levels of its restriction map which are equivalent tothose of the restriction maps of HIV-1. With the complete cellular DNAof infected cells, two types of signal are detected by the “Southernblot” method: (1) in DNA fractions having molecular weights MW ofapproximately 20 kb and more, in the case of integrated forms of thevirus, and (2) in the fractions of lower MW (9,10 kb), in the case ofthe virus not integrated in the genome.

These characteristics are highly specific to a retrovirus.

Some experiments performed with STLV-III (SIV-3) from infected cellsenabled it to be established that the simian retrovirus is relativelydistant from HIV-2 (the signal is detected exclusively under lowstringency conditions). These experiments show that the abovementionedprobes permit the specific detection of HIV-2.

(4) Subcloning of the cDNA of HIV-2 in a Bacterial Plasmid Vector:

The positive M13 clone, E 2.1, was selected and subcloned in a plasmidvector. The DNA of the recombinant M 13 (TG 130) phage E 2 was purifiedin the form of a single-stranded DNA (M−13-ROD-E2) containing the 2 kbinsert containing the 3′ portion of the HIV-2 genome (obtained fromHIV-2 ROD). This insert was transferred to plasmid pSP65, described byMelton, D. A., in 357 Nucleic Acid Res. 12:035—7056 (1984).

A second chain was constructed in vitro in the presence of the 17-merinitiator sequence (AMERSHAM), the four nucleotides A, C, T, G, and DNApolymerase I (Klenow). The EcoRI insert was excised by EcoRI digestionand purified on agarose gel, and then ligated to pSP65 which had itselfbeen digested beforehand with

EcoRI. The ligation mixture was used to transform E. coli strain DH1,and recombinants were selected on the basis of their capacity forresistance to ampicillin. The re combinants identified were cultured onLB medium (Luria medium) containing 50 μg/ml of ampilillin. Theserecombinant plasmids were purified and monitored for the presence of thecorrect inserted fragment.

One of the clones obtained, designated by the reference pSPE2, wasdeposited with the CNCM in Paris, France, under access no. I-595 on 5thSep. 1986.

The inserts derived from the cDNAs of HIV-2 and which were presentinserted in the abovementioned probe contained the nucleotide sequencewhich has been defined above, in conformity with a part of E2.

Example II

Cloning of a cDNA Complementary to the DNA Complementary to the GenomicRNA of HIV-2 Virions

HIV-2 virions were purified from 5 liters of a culture supernatant froma CEM line infected with a ROD isolate. A first strand of cDNA wassynthesized in contact with sedimented purified virus, in the presenceof an oligo(dT) initiator and employing an endogenous reaction activatedby a detergent, according to the technique described by Alizon et al.,Nature 312, 757–760 (1984). The RNA/cDNA hybrids were purified byextraction with a phenol/chloroform mixture and by precipitation withethanol. The second strand of cDNA was produced in the presence of DNApolymerase I/RNAse H, according to the method described by Gubler andHoffman ( ). The description in this paper is considered to beincorported herein by reference.

The double-stranded cDNA was provided with blunt ends in the presence ofDNA polymerase T4, using the constituents of a cDNA synthesis kitmarketed by AMERSHAM.

EcoRI adaptors (linker), marketed by PHARMACIA were attached to the endof the cDNA; the cDNA thereby modified was inserted, after digestion inthe presence of EcoRI, into a dephosphorylated phage vector M13tg130which was itself digested with EcoRI, also marketed by AMERSHAM. A cDNAband was obtained after transformation of E. coli strain TG1.Recombinant plaques (10⁴) were screened in situ on filters permittingreplicas by hybridization with the clone J19 containing the 1.5 kbHindIII fragment mentioned above, originating from HIV-1.

The filters were prehybridized in the presence of a medium containing5×SSC, 5× DENHARDT solution, 25% formaldehyde and denatured salmon spermDNA (100 micro-grammes/ml), at 37° C. for 4 hours, and then hybridizedfor 16 hours in the same buffer (Tm-42° C.) in the presence ofadditional labelled probe (4×10⁷ cpm), to provide a final hybridizationbuffer solution containing 10⁶ cpm/ml.

Washing was carried out with a 5×SSC, 0.1% SDS solution at 25° C. for 2hours (it being understood that 20×SSC corresponds to a 3 M NaCl and 0.3M sodium citrate solution). The plaques which responded positively werepurified and the M13 single-stranded DNAs were prepared and their endssequenced according to the method of Sanger et al.

Hybridization of a DNA of Cells Infected with HIV-4 and HIV-2 and ofRNAs of HIV-1, HIV-2 and of SIV Virions, Respectively, with a ProbeDerived from a Cloned cDNA of HIV-2.

The DNAs were extracted from infected CEM cells continuously producingHIV-1 and HIV-2, respectively. DNA samples of these two retroviruses,digested in some cases with 20 μg of PstI, and undigested in othercases, were subjected to electrophoresis on 0.8% agarose gel andtransferred by the “Southern” method to a nylon membrane. Small volumesof infected supernatant, taken up in an NTE buffer containing 0.1% ofSDS and having the same reverse transcriptase activity, were depositedon nitrocellulose which had been soaked beforehand in a 2×SSC solution.

A prehybridization was carried out in a solution containing 50° offormamide, 5×SSC, 5× Denhart and 100 mg/ml of denatured salmon spermDNA, for 4 hours at 42° C. A hybridization was performed in the samebuffer, to which 10% of dextran sulphate and 10⁶ cpm/ml of E2 labelledinsert (specific activity 10⁹ cpm/μg) had been added, for 16 hours at42° C. Two washings were then carried out with a 0.1×SSC, 0.1% SDSsolution for 30 min each. After exposure for 16 hours to an intensifyingscreen, the Southern spot is dehybridized in 0.4 N NaOH, neutralized,and rehybridized under the same conditions with the HIV-1 probe labelledwith 10⁹ cpm/μg.

Example III

Cloning in Phase Lambda of the Complete DNA of the HIV-2 Provirus

The DNA of CEM cells infected with HIV-2 ROD (FIG. 2, bands a and c) ispartially digested with Sau3AI. The 9–15 kb fraction was selected on a5–40% sucrose gradient and ligated to the BamHI arm of the lambda L47.1vector. The plaques (2×10⁶) obtained after in vitro packaging anddeposition on E. coli strain LA 101 were screened in situ byhybridization with the insert of the E2 cloned cDNA. Approximately 10positive clones were purified on plaques and propagated in E. coli C600recBC. The clones lambda ROD 4, ROD 27 and ROD 35 were amplified, andtheir DNAs characterized by drawing up their restriction maps and byhybridization by Southern's method with the cDNA clone of HIV-2 understringent conditions and with the gag-pol probes of HIV-1 undernon-stringent conditions.

FIG. 8 shows schematically the structures of 3 of the recombinant phagesobtained, ROD 4, ROD 27 and ROD 35.

The elongated rectangular portions of these diagrams correspond toproviral sequences originating from the DNAs of the initially infectedCEM cells, the clear portions corresponding to retroviral sequences, theshaded portions to portions of cellular DNAs and the black portions tothe LTR in the said viral sequences.

The thin lines designated by the letters L and R correspond to the armsoriginating from the Lambda L47.1 phage vector which was used for thecloning.

Some of the restriction sites have also been indicated: these are, moreespecially, the following sites: B: BamHI; E: EcoRI; H: HindIII; K: KpnIPs: PstI Pv: PvuII; S: SacI; X: XbaI.

These viral sequences have portions in common with the E2 sequence. Therelative positions of these portions, determined by hybridization withE2, are also seen in the figures.

ROD 4 is derived from a circular viral DNA. ROD 27 and ROD 35 arederived from proviruses integrated in a cellular DNA structure.

Lastly, the inserts subcloned under the conditions described above, andtheir relative positions with respect to the corresponding ROD 4, ROD 27and ROD 35 sequences, are shown in these figures.

These are, more especially, the inserts of plasmids pROD 27-5′, pROD35-3′, pROD 4.6, pROD 4.8 and pROD 4.7.

FIG. 9 is a representation of the relative intensities of thehybridization spots which were produced between ROD-4 and sub-fragments1 to 11 originating, respectively, from the different portions of asingle-stranded DNA originating from an M13 subclone containing anucleic acid derived from the whole LAV genome. The relative positionsof these various fragments with respect to the whole LAV genome(determined by sequencing) are shown at the bottom of the figure. Point12 corresponds to a control spot produced using a control DNA of thephage lambda.

The hybridization experiments in the spot transfer (dot blot) methodwere carried out under the low stringency conditions of Example IIusing, by way of a probe, the lambda ROD 4 recombinant containing thetotal (DNA of HIV-2. The washings were then carried out successivelyunder the following conditions: 2×SSC, 0.1% SDS at 25° C. (Tm-42° C.),1×SSC, 0.1% SDS at 60° C. (Tm-20° C.) and 0.1%×SSC, 0.1% SDS at 60° C.(Tm-3° C.).

The spots shown were obtained after radiographic exposure overnight.

Example IV

In Vitro Diagnostic Test for the Presence of HIV-2 Virus in a BiologicalMedium

Material and Methods

Patients

HIV-2-infected patients were recruited among individuals visiting theEgas Moniz Hospital in Lisbon either for hospitalization or forconsultation, between September 1985 and September 1986. For thisselection, all individuals of African origin or having stayed in Africaunderwent a serum test for antibodies against both HIV-1(Immunofluorescence—IFA—and/or ELISA) and HIV-2 (IFA). Only thosepatients who were proved serologically to be infected with HIV-2 wereincluded in the study.

Virus Isolation

In 12 patients, HIV isolation was attempted as previously described.Briefly, the patients' peripheral blood lymphocytes (PBL) werestimulated with PHA, co-cultured with normal human PHA-stimulated PBLsand maintained in the presence of interleukin-2 (IL-2). Cultures weremonitored for the presence of cytopathic effect (CPE) and for reversetranscriptase (RT) activity in the supernatant.

Immunofluorescence Assay (IFA)

IFA slides were prepared as follows: HIV-2-infected MOLT-4 cells werewashed twice in PBS and layered onto IFA glass slides (10⁴ cells/well),air dried and fixed with cold acetone. For IFA these cells were reactedwith a 1/10 dilution of the test serum for 45 minutes at 37° C., washed,dried, and reacted with a fluorescein-conjugated goat antihuman IgG, A,M (1/100 diluted) for 30 minutes at 37° C. After washing, cells werecounterstained in 0.006% Evans blue, mounted in 90% glycerol, 10% PBSand examined under a fluorescence microscope.

ELISA

Some patients' sera were examined for antibodies to HIV-1 using thecommercially available serum tests ELAVIA (Pasteur Diagnostics) orABBOTT.

Radioimmunoprecipitation Assay (RIPA)

HIV-1 or HIV-2 infected CEM cells were cultured in the presence of35_(S) cysteine (200 microCi/ml) for 16 hours. The supernatant wascollected, viral particles were pelleted and lysed in RIPA buffer(Tris-HCL 50 mM pH 7.5, NaCl 150 mM, EDTA 1 mM, 1% Triton X100, sodiumdeoxycholate 0.1%, SDS 0.1%). For each reaction, 50 microlitres of adilution of lysate corresponding to 10⁵ cpm was reacted with 5microlitres of test serum for 18 hours at 4° C. Immune complexes werebound to Sepharose-protein A (PHARMACIA), washed, and eluted by boilingfor 2 minutes. Eluted antigens were then analysed by SDS-polyacryl-amidegel electrophoresis and autoradiography.

Dot-Blot Hybridization

Virus isolated from patients' PBLs were pelleted and lysed in Tris-HCL10 mM pH 7.5, NaCl 10 mM, EDTA 1 mM, SDS 0.5%. One microliter of eachlysate, corresponding to 50,000 cpm of RT activity, was deposited ontonitro-cellulose. Hybridization and washing were conducted in highstringency conditions: hybridization in 6×SSC, 5× Denhart, 50%formamide, for 18 hours at 42° C.; and washing in 0.1×SSC, 0.1% SDS at65° C. W. used HIV-1 and HIV-2 probes, ³²P labelled to a specificactivity of 10⁸ cpm/microgram. The HIV-1 probe corresponds to thecomplete genome of the LAV_(BRU) isolate, and HIV-2 probe was derivedfrom a 2 kilobases cDNA clone from LAV-2_(ROD) isolate.

Results

Patient Population

Thirty patients with serological and/or virologic evidence of HIV-2infection were studied. They were 12 males and 18 females. The mean agewas 35, ranging 11–55. All patients, except one, have stayed for severalyears in West Africa: 26 were born and living in Guinea-Bissau and 2were originating from the Cape Verde Islands. One patient was an 11-yearold boy from Angola who had lived in the Cape Verde islands for severalyears. The only European in the study population was a 40 year-oldPortuguese man who had lived for 8 years in Zaire, and denied any stayin West Africa.

Clinical Presentation

Among the 30 patients 17 had AIDS, according to the CDC criteria. Themajor symptom in these patients was chronic diarrhoea, together in mostcases with a weight loss of more than 10 kilograms in a year. In 10patients the diarrhoea was found to be associated with the presence ofan intestinal opportunistic infection in 7 cases the pathogen wasIsospora belli alone, one patient had Cryptosporidium alone and 2 hadboth pathogens. In 3 cases no opportunistic intestinal pathogen wasfound. Among all 17 AIDS cases, oesophageal candidiasis was diagnosed in8. Six AIDS patients had respiratory symptoms. Pulmonary tuberculosiswas diagnoses in 2 and another unidentified mycobacterium was found inone. Two patients had pulmonary aspergillosis, one following atuberculosis. Two other AIDS patients had recurrent episodes ofpneumonitis with no pathogen identified, and one patient hadPneumocystis carinii pneumonia, which was only diagnosed post-mortem.Four of the 17 AIDS patients had Kaposi's sarcoma: in 3 cases itappeared limited to the skin, and in one patient post-mortem examinationrevealed disseminated visceral lesions. Central nervous system disorderswere found in 2 AIDS patients: one had cerebral lymphoma, and the otherhad subacute encephalitis of unknown cause.

Four patients were presenting with the symptoms of the AIDS-relatedcomplex (ARC): two had diffuse lymphadenopathy with persistent fever,one had chronic diarrhoea with weight loss, and one had recurrentepisodes of bronchopneumonia and multiple lymphadenopathies.

Among the 9 remaining patients, 6 had no symptoms that could beconsidered as related to HIV infection, one had pulmonary tuberculosisalone, one had persistent diffuse lymphadenopathy alone, and onepresented with neurologic syphillis. During the 12 months' period of thestudy 7 patients died, all presenting with AIDS.

Serological Studies

Each patient had at least one serum test for antibodies to both HIV-1and HIV-2. All patients' sera ere tested by IFA for antibodies to HIV-2,and all were positive. Among them, 21 were also tested for antibodies toHIV-2 by RIPA: all clearly precipitated the high-molecular weightenvelope glycoprotein of the virus, termed gp 140, and 16 of them alsoreacted with the major core protein p26, whereas only one reacted withanother viral core protein, termed p16.

The sera were evaluated for the presence of cross-reacting antibodies toHIV-1 using different assays. An IFA test was used in 24 sera: 12 werenegative, were weakly reactive, and 2 were positive. In ELISA, 21 weretested: 16 were negative, and 5 were positive. Finally, 11 sera weretested for antibodies to HIV-1 proteins by RIPA. Three failed to reactwith any viral protein, 2 only precipitated the pol gene product p34,and 5 reacted with the major core protein p25. Two sera reacted onlyfaintly with the envelope glycoprotein gp 110 of HIV-1. These two sera,and all sera with positive IFA or ELISA tests for antibodies to HIV-1,had a strong reactivity with the gp 140 of HIV-2 in RIPA, indicative ofinfection with HIV-2 rather than with HIV-1. Only one patient, that wedid not include in the study population, was serologically found to beinfected with HIV-1 and not with HIV-2. This patient was a 21-year-oldwoman from Central Africa (Sao Tome Islands) with AIDS.

Virus Isolation

Isolation of retroviruses from peripheral blood lymphocytes wasattempted in 12 patients. HIV was isolated in 11, according to thepresence of a typical cytopathic effect, and of a peak ofparticle-associated reverse transcriptase activity in the culturesupernatants.

All 11 isolates were identified as HIV-2 using a dot-blot hybridizationtechnique. Viral dots from 10 isolates were found to strongly hybridizein stringent conditions of hybridization and washing with a HIV-2 probederived from a cloned HIV-2 cDNA, whereas none of them hybridized with aHIV-1 probe in the same conditions. One isolate only faintly hybridizedwith the HIV-2 probe, but it failed to hybridize with the HIV-1 probe.

Immunological Evaluation

Thirteen patients were evaluated for the number of circulatinglymphocytes identified as helper T cells (CD4+) and the ratio ofhelper:supressor T cells. Among these patients, 11 had AIDS: their meanabsolute helper T cells count was 243+300/microliter and their meanhelper: suppressor ratio was 0.25+0.15. One patient, clinicallypresenting with ARC, had a number of helper T lymphocytes of240/microliter and a ratio of 0.18. Another patient, with neurologicalsyphilis and no evident HIV-related symptom, had a helper T lymphocytecount of 690/microliter and a ratio of 0.82.

DISCUSSION

In this study, we demonstrated HIV-2 infection in 30 West Africanpatients presenting either with AIDS, ARC or with no apparentHIV-related symptoms. The results nevertheless permit the conclusionthat HIV-2 must be considered to be a major aetiological agent of AIDSin West African patients. The serological and virologic profiles that weobserved indicate that HIV-2 infection was not often associated withHIV-1 infection in our patients. Despite important antigenic and geneticdifferences, HIV-1 and HIV-2 display similar tropism for CD4+ Tlymphocytes, similar cytopathic effects, similar morphology, and sharecommon immunoreactive epitopes in some of their constitutive proteins.Since all West African patients with HIV infection in this study werefound to be infected with HIV-2 and none of them with HIV-1, the newvirus HIV-2 may be the major cause of AIDS in West Africa.

The symptoms of HIV-2-related AIDS were not different from those ofHIV-1 related AIDS in Central Africa: The most common symptom waschronic diarrhoea, with important weight loss, mostly due to Isosporabelli and/or Cryptosporidium. The frequency of other opportunisticinfections, such as candidiasis, mycobacteria (including M.tuberculosis) and toxoplasmosis was found comparable to that inHIV-1-related African AIDS. Pneumocystis carinii pneumonia, a verycommon complication of AIDS in the USA and Europe, has only been foundonce in our study, and cryptococcal meningitis was not detected.

Nevertheless, the immunological abnormalities found in HIV-2-infectedAIDS patients are identical to those described in HIV-1-related AIDS.

Among the 30 patients, who all had serum antibodies reactive with HIV-2antigens, only 7 had HIV-1 specific antibodies detectable using IFA orELISA. In RIPA, all of these 7 patients had antibodies reactive with theother major core proteins p25 (HIV-1) or p26 (HIV-2), which sharestrongly immunoreactive epitopes. Five patients lacked such antibodies:all 5 had a negative HIV-1 ELISA. IFA was borderline in 3 and negativein 2. However, although some of them were not completely evaluated, wefound 9 patients with serum antibodies to the viral core protein p26 ofHIV-2 who had a weakly reactive or negative HIV-1-specific IFA and/orELISA. These findings point to the importance of including HIV-2antigens in the HIV Serum tests used in Africa and perhaps in otherareas.

A retrovirus was isolated from the peripheral lymphocytes of 11patients. In all cases, viral growth was obtained within 2 weeks,characterized by the presence of reverse transcriptase activity in theculture supernatant and of cytopathic effect. However, this cytopathiceffect appeared to vary in importance from one isolate to another: someisolates provided numerous large-sized syncytia together with importantcell lysis, whereas others exhibited only few syncytia of limited size,and affected poorly the viability of the culture.

RNA from all but one isolate was found to clearly hybridize in highstringency conditions with a prove derived from a HIV-2 cDNA clone,representing the 3 end of the genome. None hybridized with a HIV-1 provein the same conditions. This demonstrates that the isolates infectingthese patients all belonged to the same type of virus. One isolate onlypoorly hybridized with HIV-1. This virus, however, was isolated from apatient with serum antibodies reacting with all the antigens of HIV-2 inRIPA.

This invention relates generally, in addition to HIV-2 virus itsvariants, to any equivalent virus which is infectious for man andpossesses immunological characteristics specific to HIV-2. The inventionrelates generally to any virus which, in addition to the propertiespossessed by the HIV-2 viruses deposited with the CNCM, also possessesthe characteristics which follow.

The preferred target for the HIV-2 retrovirus consists of human Leu 3cells (or T4 lymphocytes) and and “immortalized” cells derived fromthese T4 lymphocytes, for example cells of the HUT 78 lines dealt within the context of this patent application. In other words, it has aspecific tropism for these cells. It can be cultured in permanent linesof the HUT, CEM, MOLT or similar type, expressing the T4 protein. It isnot infectious for T8 lymphocytes. It is cytotoxic for the human T4lymphocytes. The cytopathogenic nature of HIV-2 viruses with respect toT4 lymphocytes manifests itself, in particular, by the appearance ofmultinucleated cells. It has a reverse transcriptase activity whichrequires the presence of Mg²⁺ ions and has a strong affinity forpolyadenylate oligodeoxythymidylate (poly(A)-oligo(dT) 12–18). It has adensity of approximately 1.16 in a sucrose gradient. It has a meandiameter of 140 nanometers and a core having a mean diameter of 41nanometers. The lysates of this virus contain a p26 protein which doesnot cross immunologically with the p24 protein of HTLV-1 virus orHTLV-II virus. These p26 proteins hence have a molecular weight which isslightly higher (by approximately 1000) than the corresponding p25proteins of HIV-1 and slightly lower (again, of the order ofapproximately 1000 lower) than the corresponding p27 proteins of theSIV. The lysates of HIV-2 contain, in addition, a p16 protein which isnot immunologically recognized by the p19 protein of HTLV-1 or ofHTLV-II in RIPA (abbreviation for radioimmunoprecipitation assay)experiments. They contain, in addition, an envelope glycoprotein havinga molecular weight of the order of 130,000–140,000, which does not crossimmunologically with the gp 110 of HIV-1 but which, on the other hand,crosses immunologically with the gp 140 envelope glyco-protein ofSTLV-III. These lysates also contain proteins or glycoproteins which canbe labelled with (³⁵S)cysteine, having molecular weights, respectively,of the order of 36,000 and 42,000–45,000. The genomic RNA of HIV-2 doesnot hybridize with the genomic RNA of HIV-1 under stringent conditions.Under non-stringent conditions, it does not hybridize with nucleotidesequence derived from HIV-1 and containing the env gene and the LTRadjacent to it. In particular, it does not hybridize with the nucleotidesequence 5290–9130 of HIV-1, nor with sequences of the pol region of theHIV-1 genome, in particular with the nucleotide sequence 2170–2240.under non-stringent conditions, it hybridizes weakly with nucleotidesequences of the HIV-1 region, in particular the nucleotide sequences990–1070 and 990–1260.

It should be noted that any retrovirus which is infectious for man,capable of inducing one of the forms of AIDS, having the abovementionedessential properties and whose genomic RNA is capable of hibridizingunder stringent conditions with those HIV-2 viral strains deposited withthe CNCM (or with a cDNA or cDNA fragment derived from these genomicRNAs) is to be considered to be an equivalent of HIV-2.

The invention also relates to each of the antigens, in particularproteins and glycoproteins in the purified state, such as may beobtained from HIV-2. Reference to “purified” proteins or glycoproteinsimplies that these proteins or glycoproteins lead, respectively, only tosingle bands in polyacrylamide gel electrophoresis, in particular underthe experimental conditions which have been described above. Anysuitable method of separation and/or purification for obtaning each ofthese can be used. By way of example of a technique which can beemployed, that describes by R. C. MONTELARO et al., J. of Virology, June1982, pp. 1029–1038, will be mentioned.

The invention relates generally to all antigens, in particular proteins,glycoproteins or polypeptides, originating from an HIV-2 and havingimmunological properties equivalent to those of these antigeniccompounds of HIV-2. Two antigens are said to be “equivalent”, in thecontext of this account, inasmuch as they are recognized by the sameantibodies, in particular antibodies which can be isolated from a serumobtained from a patient who has been infected with an HIV-2, or inasmuchas they meet the conditions for “immunological equivalence” statedbelow.

Among equivalent polypeptides, proteins or glycoproteins, there must beincluded fragments of the above antigens (or peptides reconstituted bychemical synthesis), inasmuch as they give rise to immunologicalcross-reactions with the antigens from which they are derived. In otherwords, the invention relates to any polypeptide which has, in commonwith abovementioned antigens, identical or similar epitopes capable ofbeing recognized by the same antibodies. Belonging to this latter typeof polypeptides are the products of expression of correspondingsequences of the DNAs which code for the corresponding polypeptidesequences.

The HIV-2 virus has proved to be usable as a source of antigens fordetecting antibodies in all people who have come into contact with theHIV-2 virus.

The invention relates generally to any composition which can be used forthe diagnosis of the presence in a biological fluid serum, in particularof people who have come into contact with HIV-2, of antibodies againstat least one of the antigens of HIV-2. This composition can be appliedto the selective diagnosis of the corresponding variety of AIDS,employing diagnostic techniques such as those described in the Europeanpatent application cited above, except that εχτθαψτσ, lysates orpurified antigens of HIV-2 are used instead of those of HIV-1. In thisconnection, the invention relates more especially to compositionscontaining at least one of the proteins p12, p16, p26, which are theinternal proteins, or p36 or gp 140. By way of examples of compositions,those which simultaneously contain the following will be mentioned.

p26 and gp 36 p26, p36 and gp 140, p12, p16 and p26, p16, p26 and gp140, etc.

it is self-evident that these compositions signify only examples. Inparticular, the invention relates to the viral extracts or lysatescontaining this group of proteins and/or glycoproteins or all fractionsfrom which one or more of the abovementioned proteins or glycoproteinshas been separated beforehand.

The invention also relates to compositions containing a combination ofproteins and/or glycoproteins of an HIV-2 with proteins and/orglycoproteins of an HIV-1, for example:

either core proteins of HIV-1 and HIV-2, in particular the p25 of anHIV-1 and p26 of an HIV-2, or alternatively the p18 of an HIV-1 and thep16 of an HIV-2,

or envelope glycoproteins of an HIV-1 and envelope glycoproteins of anHIV-2, in particular the gp 110 of HIV-1 and the gp 140 of HIV-2, oralternatively the p42 of HIV-1 and the p36 or p42–45 of HIV-2,

or, of course, mixtures of proteins and/or glycoproteins of an HIV-1 andproteins and/or glycoproteins of and HIV-2.

Such compositions, used for diagnosis, consequently make possibleprocedures for diagnosis of AIDS or of the symptoms which are associatedwith it, which extend over a wider spectrum of the aetiological agentsresponsible. It goes without saying that the use for diagnosticprocedures of compositions which contain only proteins and/orglycoproteins of HIV-2 is nevertheless useful for more selectivediagnosis of the category of retrovirus which can be held responsiblefor the disease.

The invention also relates to the DNAs or DNA fragments, more especiallycloned DNAs and DNA fragments, obtained from the RNA and cDNAs derivedfrom the RNA of the HIV-2 retrovirus. The invention also relatesespecially to all equivalent DNAs, in particular any DNA possessingsequence homologies with the DNA of HIV-2, especially with the sequenceswhich code for the env and pol regions of the strain of HIV-2 depositedwith the CNCM, equal at least to 50%, preferably to 70% and still moreadvantageously to 90%. It will also be stated generally that theinvention relates to any equivalent DNA (or RNA) capable of hybridizingwith the DNA or RNA of HIV-2 in the “spot blot” technique, undernon-stringent conditions as defined above.

The invention likewise relates to the sera capable of being produced inanimals by inoculating the latter with HIV-2. The invention hencerelates more especially to the polyclonal antibodies directed morespecifically against each of the antigens, in particular proteins orglycoproteins, of the virus. It also relates to the monoclonalantibodies which can be produced by traditional techniques, thesemonoclonal antibodies being directed, respectively, more Specificallyagainst the different proteins of HIV-2.

These polyclonal or monoclonal antibodies can be used in differentapplications. Their use for neutralizing the corresponding proteins, oreven inhibiting the infectivity of the whole virus, will mainly bementioned. They can also be used, for example, for demonstrating theviral antigens in biological preparations or for carrying out proceduresfor purification of the corresponding proteins and/or glycoproteins, forexample by using them in affinity chromatography columns.

It is understood that, in general, the available technical literature(in particular that for which the bibliographic references are in thecontext of the present description) in respect of HIV-1 and the virusdesignated HTLV-III is to be considered to be incorporated herein byreference, inasmuch as the techniques described in this literature areapplied under similar conditions to the isolation of HIV-2 virus or ofthe equivalent viruses, and to the production from these viruses oftheir different constituents (in particular proteins, glycoproteins,polypeptides and nucleid acids). Use can also be made of the teachingsof this technical literature as regards the application of the differentconstituents in question, in particular to diagnostic procedures of thecorresponding forms of LAS or AIDS.

The present invention relates more especially to a method for in vitrodiagnosis of AIDS, which comprises bringing a serum or anotherbiological medium originating from a patient who is the subject of thediagnosis into contact with a composition containing at least one of theproteins or glycoproteins of HIV-2, or alternatively an extract orlysate of the virus, and the detection of the immunological reaction.Examples of such compositions have been stated above.

Preferred methods involved, for example, immunoenzymatic reactions ofthe ELISA or immunofluorescence type. The titrations can be measurementsby direct or indirect immunoflueorescence, by direct or indirectimmunoenzymatic assays.

Thus, the present invention also relates to extracts of virus (either anextract of one or more HIV-2 viruses alone, or a mixture of extractsoriginating from one or more HIV-2 viruses, on the one hand, and one ormore HIV-1 viruses, on the other hand), these extracts being labelled.Any suitable type of label can be used enzymatic, fluorescent,radioactive and the like.

Such titrations comprise, for example:

the deposition of specified amounts of the extract or of the compositionreferred to according to the present invention in the wells of amicrotitration plate;

introduction into these wells of increasing dilutions of serumprincipally containing the antibodies whose presence is to be detectedin vitro;

the incubation of the microtitration plate;

careful washing of the microtitration plate with a suitable buffer;

the introduction into the wells of the microtitration plate of labelledantibodies specific for human immunoglobulins, the labelling beingcarried out with an enzyme chosen from those which are capable ofhydrolysing a substrate in such a way that the latter then undergoes amodification of its absorption of radiation, at least in particularwavelength band, and

the detection, preferably in comparative fashion relative to a control,of the extent of hydrolysis of the substrate, as a measurement of thepotential risks or of the effective presence of the disease.

The present invention also relates to outfits or kits for the abovediagnosis, which comprise:

an extract or a more highly purified fraction of the types of virusstated above, this extract or fraction being labelled, for exampleradioactively, enzymatically or by immunofluorescence;

anti-(human immunoglobulins) or a protein A (advantageously, bound to asupport which is insoluble in water, such as agarose beads);

an extract of lymphocytes obtained from a person in good health;

buffers and, where appropriate, substrates for the visualization of thelabelling.

It emerges from the foregoing that the invention relates to thediagnosis of HIV-2 virus, or of a variant, as a result of the use of theprobes described above, in a method employing different stages recordedbelow, these stages being arranged specifically to bring out thecharacteristic properties of the HIV-2 virus.

The invention naturally also relates to the use of the cDNAs or theirfragments (or recombinants containing them) as probes for the diagnosisof the presence or absence of HIV-2 virus in samples of serum or ofother biological fluids or tissues obtained from patients suspected ofbeing carriers of the HIV-2 virus.

These probes are preferably also labelled radioactively, enzymatic,fluorescent labels, and the like). Especially advantageous probes forcarrying out the method for diagnosis of the HIV-2 virus, or of avariant of HIV-2, can be characterized in that they comprise all or afraction of the cDNA complementary to the genome of the HIV-2 virus, oralternatively, in particular, the fragments present in the variousclones identified above. There will be mentioned, more especially, afraction of the cDNA of HIV-2 present in the clone E2, more especiallythe sequence of the 3′ end (LTR) and/or of the 5′ end of the HIVsequence of the above-mentioned clone E2, or alternatively the cDNAcontaining the env region, of the cDNA of the HIV-2 virus.

The probes employed in this method for diagnosis of the HIV-2 virus andin the diagnostic kits are in no way limited to the probes describedabove. They comprise, on the contrary, all the nucleotide sequencesoriginating from the genome of the HIV-2 virus, of a variant of HIV-2 orof a structurally related virus, inasmuch as they enabled antibodiesdirected against an HIV-2 to be detected in biological fluids of peoplecapable of developing one of the forms of AIDS. Naturally, the use ofnucleotide sequences originating from an HIV-2 which is initiallyinfectious for man is nevertheless, preferred.

The detection can be carried out in all manners known per se, inparticular, by bringing these probes into contact either with thenucleic acids obtained from cells present in these sera or otherbiological media, for example, cerebrospinal fluids, saliva, and thelike, or with these media themselves inasmuch us their nucleic acidshave been rendered accessible to hybridization with these probes, thisbeing under conditions which permit hybridization between these probesand these nucleic acids, and by detection of the hybridization which maybe produced. The above-mentioned diagnosis, involving hybridizationreactions, can also be carried out using mixtures of probes originating,respectively, from an HIV-1 and an HIV-2, insofar as it is unnecessaryto differentiate between the type of HIV virus sought.

In general, the method for diagnosis of the presence or absence of theHIV-2 virus or a variant, in samples of sera or other fluids or tissuesobtained from patients suspected of being carriers of the HIV-2 virus,comprises the following stages:

1) the manufacture of a labelled probe,

2) at least one hybridization stage performed under stringentconditions, by bringing the DNA of cells in the sample from the suspectpatient into contact with the said labelled probe or a suitablemembrane,

3) washing of the said membrane with a solution which provides for theretention of these stringent conditions for the hybridization, and

4) the detection of the presence or absence of the HIV-2 virus by animmunodetection method.

In another preferred embodiment of the method according to theinvention, the above-mentioned hybridization is performed undernon-stringent conditions and the washing of the membrane is carried outunder conditions adapted to those for the hybridization.

The invention relates in particular to HIV-2 viruses, characterized inthat their viral RNA corresponds with a cDNA whose GAG and ENV genescomprise respectively the nucleotidic sequences which follow.

They result from the sequencing of corresponding regions of cDNAcorresponding to the genome HIV-2 Rod. They are in correspondance withthe amino-acids that they code.

MetGlyAlaArgAsnSerValLeuArgGlyLysLysAlaAspGluATGGGCGCGAGAAACTCCGTCTTGAGAGGGAAAAAAGCAGATGAA         *         *         *         *LeuGluArgIleArgLeuArgProGlyGlyLysLysLtsTyrArgTTAGAAAGAATCAGGTTACGGCCCGGCGGAAAGAAAAAGTACAGG    *         *         *         *         *LeuLysHisIleValTrpAlaAlaAsnLysLeuAspArgPheGlyCTAAAACATATTGTGTGGGCAGCGAATAAATTGGACAGATTCGGA       100         *         *         *LeuAlaGluSerLeuLeuGluSerLysGluGlyCysGlnLysIleTTAGCAGAGAGCCTGTTGGAGTCAAAAGAGGGTTGTCAAAAAATT    *         *         *         *         *LeuThrValLeuAspProMetValProThrGlySerGluAsnLeuCTTACAGTTTTAGATCCAATGGTACCGACAGGTTCAGAAAATTTA         *       200         *         *LysSerLeuPheAsnThrValCysValIleTrpCysIleEisAlaAAAAGTCTTTTTAATACTGTCTGCGTCATTTGGTGCATACACGCA    *         *         *         *         *GluGluLysValLysAspThrGluGlyAlaLysGlnIleValArgGAACAGAAAGTGAAAGATACTGAAGGAGCAAAACAAATAGTGCCG         *         *       300         *ArgHisLeuValAlaGluThrGlyThrAlaGluLysMetProSerAGACATCTAGTGGCAGAAACAGGAACTGCAGAGAAAATGCCAAGC    *         *         *         *         *ThrSerArgProThrAlaProSerSerGluLysGlyGlyAsnTyrACAAGTAGACCAACAGCACCATCTAGCGAGAAGGGAGGAAATTAC         *         *         *       400ProValGlnHisValGlyGlyAsnTyrThrHisIleProLeuSerCCAGTGCAACATGTAGGCGGCAACTACACCCATATACCGCTGAGT    *         *         *         *         *ProArgThrLeuAsnAlaTrpValLysLeuValGluGluLysLysCCCCGAACCCTAAATGCCTGGGTAAAATTAGTAGAGGAAAAAAAG         *         *         *         *PheGlyAlaGluValValProGlyPheGlnAlaLeuSerGluGlyTTCGGGGCAGAAGTAGTGCCAGCATTTCAGGCACTCTCAGAAGGC  500         *         *         *         *CysThrProTyrAspIleAsnGlnMetLeuAsnCysValGlyAspTGCACGCCCTATGATATCAACCAAATGCTTAATTGTGTGGGCGAC         *         *         *         *HisGlnAlaAlaMetGluIleIleArgGluIleIleAsnGluGluCATCAAGCAGCCATGCAGATAATCAGGGAGATTATCAATGAGGAA    *       600         *         *         *AlaAlaGluTrpAspValGlnHisProIleProGlyProLeuProGCAGCAGAATGGGATGTGCAACATCCAATACCAGGCCCCTTACCA         *         *         *         *AlaGlyGluLeuArgGluProArgGlySerAspIleAlaGlyThrGCGGGGCAGCTTAGAGAGCCAAGGGGATCTGACATAGCAGGGACA    *         *       700         *         *ThrSerThrValGluGluGlnIleGlnTrpMetPheArgProGluACAAGCACAGTAGAAGAACAGATCCAGTGGATGTTTAGGCCACAAAsnProValProValGlyAsnIleTyrArgArgTrpIleGlnIleAATCCTGTACCAGTAGGAAACATCTATAGAAGATCCATCCAGATA    *         *         *       800         *GlyLeuGlnLysCysValArgMetTyrAsnProThrAsnIleLeuGGATTGCAGAAGTGTGTCAGGATGTACAACCCGACCAACATCCTA         *         *         *         *AspIleLysGlnGlyProLysGluProPheGlnSerTyrValAspGACATAAAACAGGGACCAAAGGAGCCGTTCCAAAGCTATGTAGAT    *         *         *         *       900ArgPheTyrLysSerLeuArgAlaGluGlnThrAspProAlaValAGATTCTACAAAAGCTTGAGGGCAGAACAAACAGATCCAGCAGTG         *         *         *         *LysAsnTrpMetThrGlnThrLeuLeuValGlnAsnAlaAsnProAAGAATTGGATGACCCAAACACTGCTAGTACAAAATGCCAACCCA    *         *         *         *         *AspCysLysLeuValLeuLysGlyLeuGlyMetAsnProThrLeuGACTGTAAATTAGTGCTAAAAGGACTAGGGATGAACCCTACCTTA      1000         *         *         *GluGluMetLeuThrAlaCysGlnGlyValGlyGlyProGlyGlnGAAGAGATGCTGACCGCCTGTCAGGGGGTAGGTGGGCCAGGCCAG    *         *         *         *         *LysAlaArgLeuMetAlaGluAlaLeuLysGluValIleGlyProAAAGCTAGATTAATGGCAGAGGCCCTGAAAGAGGTCATAGGACCT         *      1100         *         *AlaProIleProPheAlaAlaAlaGlnGlnArgLysAlaPheLysGCCCCTATCCCATTCGCAGCAGCCCAGCAGAGAAAGGCATTTAAA    *         *         *         *         *CysTrpAsnCysGlyLysGluGlyHisSerAlaArgGluCysArgTGCTGGAACTGTGGAAAGGAAGGGCACTCGGCAAGACAATGCCGA         *         *      1200         *AlaProArgArgGlnGlyCysTrpLysCysGlyLysProGlyHisGCACCTAGAAGGCAGGGCTGCTGGAAGTGTGGTAAGCCAGGACAC    *         *         *         *         *IleMetThrAsnCysProAspArgGlnAlaGlyPheLeuGlyLeuATCATGACAAACTGCCCAGATAGACAGGCAGGTTTTTTAGGACTG         *         *         *      1300GlyProTrpGlyLysLysProArgAsnPheProValAlaGlnValGGCCCTTGGGGAAAGAAGCCCCGCAACTTCCCCGTGGCCCAAGTT    *         *         *         *         *ProGlnGlyLeuThrProThrAlaProProValAspProAlaValCCGCAGGGGCTGACACCAACAGCACCCCCAGTGGATCCAGCAGTG         *         *         *         *AspLeuLeuGluLysTyrMetGlnGlnGlyLysArgGlnArgGluGATCTACTGGAGAAATATATGCAGCAAGGGAAAAGACAGAGAGAG 1400         *         *         *         *GlnArgGluArgProTyrLysGluValThrGluAspLeuLeuHisCAGAGAGAGAGACCATACAACCAACTGACAGAGCACTTACTGCAC         *         *         *         *LeuGluGlnGlyGluThrProTyrArgGluProProThrGluAspCTCGAGCAGGGGGAGACACCATACAGGGAGCCACCAACAGAGGAC    *      1500         *         *         *LeuLeuHisLeuAsnSerLeuPheGlyLysAspGluTTGCTGCACCTCAATTCTCTCTTTGGAAAAGACCAG          *         *         *MetMetAsnGlnLeuLeuIleAlaIleLeuLeuAlaSerAlaCysATGATGAATCAGCTGCTTATTGCCATTTTATTAGCTAGTGCTTGC         *         *         *         *LeuValTyrCysThrGlnTyrValThrValPheTyrGlyValProTTAGTATATTGCACCCAATATGTAACTGTTTTCTATGGCGTACCC    *         *         *         *         *ThrTrpLysAsnAlaThrIleProLeuPheCysAlaThrArgAsnACGTGGAAAAATGCAACCATTCCCCTCTTTTGTGCAACCAGAAAT       100         *         *         *ArgAspThrTrpGlyThrIleGlnCysLeuProAspAsnAspAspAGGGATACTTGGGGAACCATACAGTGCTTGCCTGACAATGATGAT    *         *         *         *         *TyrGlnGluIleThrLeuAsnValThrGluAlaPheAspAlaTrpTATCAGGAAATAACTTTGAATGTAACAGAGGCTTTTGATGCATGG         *       200         *         *AsnAsnThrValThrGluGlnAlaIleGluAspValTrpHisLeuAATAATACAGTAACAGAACAAGCAATAGAAGATGTCTGGCATCTA    *         *         *         *         *PheGluThrSerIleLysProCysValLysLeuThrProLeuCysTTCGAGACATCAATAAAACCATGTGTCAAACTAACACCTTTATGT         *         *       300         *ValAlaMetLysCysSerSerThrGluSerSerThrGlyAsnAsnGTAGCAATGAAATGCAGCAGCACACAGAGCAGCACAGGGAACAAC    *         *         *         *         *ThrThrSerLysSerThrSerThrThrThrThrThrProThrAspACAACCTCAAAGAGCACAAGCACAACCACAACCACACCCACAGAC         *         *         *       400GlnGluGlnGluIleSerGluAspThrProCysAlaArgAlaAspCAGGAGCAAGAGATAAGTGAGGATACTCCATGCGCACGCGCAGAC    *         *         *         *         *AsnCysSerGlyLeuGlyGluGluGluThrIleAsnCysGlnPheAACTGCTCAGGATTGGGAGAGGAAGAAACGATCAATTGCCAGTTC         *         *         *         *AsnMetThrGlyLeuGluArgAspLysLysLysGlnTyrAsnGluAATATGACAGGATTAGAAAGAGATAAGAAAAAACAGTATAATCAA  500         *         *         *         *ThrTrpTyrSerLysAspValValCysGluThrAsnAsnSerThrACATGGTACTCAAAAGATGTGGTTTGTGAGACAAATAATAGCACA         *         *         *         *AsnGlnThrGlnCysTyrMetAsnEisCysAsnThrSerValIleAATCAGACCCAGTGTTACATGAACCATTGCAACACATCAGTCATC    *       600         *         *         *ThrGluSerCysAspLysHisTyrTrpAspAlaIleArgPheArgACAGAATCATGTGACAAGCACTATTGGGATGCTATAAGGTTTAGA         *         *         *         *TyrCysAlaProProGlyTyrAlaLeuLeuArgCysAsnAspThrTACTGTGCACCACCGGGTTATGCCCTATTAAGATGTAATGATACC    *         *       700         *         *AsnTyrSerGlyPheAlaProAsnCysSerLysValValAlaSerAATTATTCAGGCTTTGCACCCAACTGTTCTAAAGTAGTAGCTTCT         *         *         *         *ThrCysThrArgMetMetGluThrGlnThrSerThrTrpPheGlyACATGCACCAGGATGATGGAAACGCAAACTTCCACATGGTTTGGC    *         *         *       800         *PheAsnGlyThrArgAlaGluAsnArgThrTyrIleTyrTrpHisTTTAATGGCACTAGAGCAGAGAATAGAACATATATCTATTGGCAT         *         *         *         *GlyArgAspAsnArgThrIleIleSerLeuAsnLysTyrTyrAsnGGCAGAGATAATAGAACTATCATCAGCTTAAACAAATATTATAAT    *         *         *         *       900LeuSerLeuHisCysLysArgProGlyAsnLysThrValLysGlnCTCAGTTTGCATTGTAAGAGGCCAGGGAATAAGACAGTGAAACAA         *         *         *         *IleMetLeuMetSerGlyHisValPheHisSerEisTyrGlnProATAATGCTTATGTCAGGACATGTGTTTCACTCCCACTACCAGCCG    *         *         *         *         *IleAsnLysArgProArgGlnAlaTrpCysTrpPheLysGlyLysATCAATAAAAGACCCAGACAAGCATGGTGCTGGTTCAAAGGCAAA      1000         *         *         *TrpLysAspAlaMetGluGluValLysThrLeuAlaLysEisProTGGAAAGACGCCATGCAGGAGGTGAAGACCCTTGCAAAACATCCC    *         *         *         *         *ArgTyrArgGlyThrAsnAspThrArgAsnIleSerPheAlaAlaAGGTATAGAGGAACCAATGACACAAGGAATATTAGCTTTGCAGCG         *      1100         *         *ProGlyLysGlySerAspProGluValAlaTyrMetTrpThrAsnCCAGGAAAAGGCTCAGACCCAGAAGTAGCATACATGTGGACTAAC    *         *         *         *         *CysArgGlyGluPheLeuTyrCysAsuMetThrTrpPheLeuAsuTGCAGAGGAGAGTTTCTCTACTGCAACATGACTTGGTTCCTCAAT         *         *      1200         *TrpIleGluAsnLysThrHisArgAsnTyrAlaProCysHisIleTGGATAGAGAATAAGACACACCGCAATTATGCACCGTGCCATATA    *         *         *         *         *LysGlnIleIleAsnThrTrpEisLysValGlyArgAsnValTyrAAGCAAATAATTAACACATGGCATAAGGTAGGGAGAAATGTATAT         *         *         *      1300LeuProProArgGluGlyGluLeuSerCysAsnSerThrValThrTTGCCTCCCAGGGAACCGGAGCTGTCCTGCAACTCAACAGTAACC    *         *         *         *         *SerIleIleAlaAsnIleAspTrpGlnAsnAsnAsnGlnThrAsnAGCATAATTGCTAACATTGACTGGCAAAACAATAATCAGACAAAC         *         *         *         *IleThrPheSerAlaGluValAlaGluLeuTyrArgLeuGluLeuATTACCTTTAGTGCACACGTGGCACAACTATACACATTGGAGTTG 1400         *         *         *         *GlyAspTyrLysLeuValGluIleThrProIleGlyPheAlaProGGAGATTATAAATTGGTAGAAATAACACCAATTGGCTTCGCACCTThrLysGluLysArgTyrSerSerAlaHisGlyArgHisThrArgACAAAAGAAAAAAGATACTCCTCTGCTCACGGGAGACATACAAGA    *      1500         *         *         *GlyValPheValLeuGlyPheLeuGlyPheLeuAlaThrAlaGlyGGTGTGTTCGTGCTAGGGTTCTTGGGTTTTCTCGCAACAGCAGGT         *         *         *         *SerAlaMetGlyAlaArgAlaSerLeuThrValSerAlaGlnSerTCTGCAATGGGCGCTCGAGCGTCCCTGACCGTGTCGGCTCAGTCC    *         *      1600         *         *ArgThrLeuLeuAlaGlyIleValGlnGlnGlnGlnGlnLeuLeuCGGACTTTACTGGCCGGGATAGTGCAGCAACAGCAACAGCTGTTG         *         *         *         *AspValValLysArgGlnGlnGluLeuLeuArgLeuThrValTrpGACGTGGTCAAGAGACAACAAGAACTGTTGCGACTGACCGTCTGG    *         *         *      1700         *GlyThrLysAsnLeuGlnAlaArgValThrAlaIleGluLysTyrGGAACGAAAAACCTCCAGGCAAGAGTCACTGCTATAGAGAAGTAC         *         *         *         *LeuGlnAspGlnAlaArgLeuAsnSerTrpGlyCysAlaPheArgCTACAGGACCAGGCGCGGCTAAATTCATGGGGATGTGCGTTTACA    *         *         *         *      1800GlnValCysHisThrThrValProTrpValAsnAspSerLeuAlaCAAGTCTGCCACACTACTGTACCATGGGTTAATGATTCCTTACCA         *         *         *         *ProAspTrpAspAsnMetThrTrpGlnGluTrpGluLysGlnValCCTGACTGGGACAATATGACGTGGCAGGAATGGGAAAAACAAGTC    *         *         *         *         *ArgTyrLeuGluAlaAsnIleSerLysSerLeuGluGlnAlaGlnCGCTACCTGGAGGCAAATATCAGTAAAAGTTTAGAACAGGCACAA      1900         *         *         *IleGlnGlnGluLysAsnMetTyrGluLeuGlnLysLeuAsnSerATTCAGCAAGAGAAAAATATGTATGAACTACAAAAATTAAATAGC    *         *         *         *         *TrpAspIlePheGlyAsnTrpPheAspLeuThrSerTrpValLysTGCGATATTTTTGGCAATTGGTTTGACTTAACCTCCTGGGTCAAG         *      2000         *         *TyrIleGlnTyrGlyValLeuIleIleValAlaValIleAlaLeuTATATTCAATATGGAGTGCTTATAATAGTAGCAGTAATAGCTTTA    *         *         *         *         *ArgIleValIleTyrValValGlnMetLeuSerArgLeuArgLysAGAATAGTGATATATGTAGTACAAATGTTAAGTAGGCTTAGAAAG         *         *      2100         *GlyTyrArgProValPheSerSerProProGlyTyrIleGlnGlnGGCTATAGGCCTGTTTTCTCTTCCCCCCCCGGTTATATCCAACAGIleEisIleHisLysAspArgGlyGlnProAlaAsnGluGluThrATCCATATCCACAAGGACCGGGGACAGCCAGCCAACGAAGAAACA         *         *         *      2200GluGluAspGlyGlySerAsnGlyGlyAspArgTyrTrpProTrpGAAGAAGACGGTGGAAGCAACGGTGGAGACAGATACTGGCCCTGG    *         *         *         *         *ProIleAlaTyrIleHisPheLeuIleArgGlnLeuIleArgLeuGCGATAGCATATATACATTTCCTGATCCGCCAGCTGATTCGCCTC         *         *         *         *LeuThrArgLeuTyrSerIleCysArgAspLeuLeuSerArgSerTTCACCAGACTATACAGCATCTGCAGGGACTTACTATCCAGGAGC 2300         *         *         *         *PheLeuThrLeuGluLeuIleTyrGlnAsnLeuArgAspTrpLeuTTCCTGACCCTCCAACTCATCTACCAGAATCTCAGAGACTGGCTG         *         *         *         *ArgLeuArgThrAlaPheLeuGlnTyrGlyCysGluTrpIleGluAGACTTAGAACAGCCTTCTTGCAATATGGGTGCGAGTGGATCCAA    *      2400         *         *         *GluAlaPheGlnAlaAlaAlaArgAlaThrArgGluThrLeuAlaGAAGCATTCCAGGCCGCCGCGAGGGCTACAAGAGAGACTCTTGCG         *         *         *         *GlyAlaCysArgGlyLeuTrpArgValLeuGluArgIleGlyArgGGCGCGTGCACGGGCTTGTGGAGGGTATTGGAACGAATCGGGAGG    *         *      2500         *         *GlyIleLeuAlaValProArgArgIleArgGlnGlyAlaGluIleGGAATACTCGCGGTTCCAAGAAGGATCAGACAGGGAGCAGAAATC         *         *         *         *AlaLeuLeu***GlyThrAlaValSerAlaGlyArgLeuTyrGluGCCCTCCTGTCAGGGACGGCAGTATCAGCAGGGAGACTTTATGAA    *         *         *      2600         *TyrSerMetGluGlyProSerSerArgLysGlyGluLysPheValTACTCCATGGAAGGACCCAGCAGCAGAAAGGGAGAAAAATTTGTA         *         *         *         * GlnAlaThrLysTyrGlyCAGGCAACAAAATATGGA     *         *

As already stated above, the invention naturally results to all HIV-2viruses whose RNAs possess similar characteristics, particularly GAG andENV regions which comprise sequences having nucleotidic sequencehomologies of at least 50%, preferably 70% and still more advantageously90% with the corresponding GAG and ENV sequences of HIV-2 Rod.

The invention relates more particularly to the cDNA fragments whichcode, respectively, for the p16, p26 and p12 whose structures are alsoincluded in GAGRODN. In particular, it relates to the sequencesextending:

from nucleotide 1 to nucleotide 405 (coding for p16);

from nucleotide 406 to nucleotide 1 155 (coding for p26); and

from nucleotide 1 156 to nucleotide 1 566 (coding for p12).

It also relates particularly to the cDNA fragment which codes for thegp140 included in ENVR and extending from the nucleotide 1 to thenucleotide 2 574.

The invention also relates to nucleotide sequences which distinguishfrom the preceding ones by nucleotide substitutions taking advantage ofthe degeneracy of the genetic code, as long as the substitutions do notinvolve a modification of the amino-acid sequences encoded by saidnucleotide sequences.

Likewise the invention concerns proteins or glycoproteins whoseamino-acid sequences correspond to those which are indicated in thepreceding pages, as well as equivalent peptides, i.e. peptides whichresult from the preceding pages by addition, substitution or dilution ofamino-acids which do not affect the overall immunological properties ofsaid peptides.

The invention concerns especially the envelope glycoprotein whichexhibits the amino-acid sequence encoded by ENVRN.

The invention also relates to an immunogenic composition characterizedin that it comprises dosage units of envelope antigen, particularly thegp140 of the HIV-2 virus, such as to enable the administration of dosageunits from 10 to 500, particularly from 50 to 100 mg/kg of body weight.

Finally the invention concerns a process for producing any of the aboveindicated proteins (p12, p16 or p26) or of the protein having thestructure of gp140, or of any determined part of said proteins, whichprocess comprises inserting the corresponding nucleic acid sequence in avector capable of transforming a suitably chosen cellular host and topermit the expression of an insert contained in said vector,transforming said chosen host by said vector which contains said nucleicsequence, culturing the cellular host transformed by said modifiedvector, recovering and purifying the protein expressed.

The techniques disclosed in European patent application 85 905513.9filed on Oct. 18, 1985 for the production of peptides or proteinsconsisting of expression products of nucleic acid sequences derived ofthe genome of HIV-1 are also applicable to the production of the abovesaid peptides or proteins derived of HIV-2. The description of thisEuropean application is incorporated herein by reference, particularlyas concerns the techniques.

As an indication, molecular weights (MP) of HIV-2 proteins are given incomparison with those of HIV-1.

MP of HIV-2 proteins MP of HIV-1 proteins kd kd entire gag 58, 3 entiregag 55, 8 p 16 15 p 18 14, 9 p 26 27, 6 p 12 15, 8 env 98, 6 env 97, 4external env 57, 4 Transmembrane env 41, 2

HIV-2 Mir and HIV-2ROD have also been deposited in the “NationalCollection of Animal Cell Cultures” (ECACC) in Salisbury (Great-Britain)on Jan. 9, 1987, under accessing numbers 87 011001 and 87 011002respectively.

Moreover, plasmids pROD35 and pROD27.5 have been deposited at the“National Collection of Industrial Bacteria” (NCIB) in Aberdeen(Great-Britain) on Jan. 9, 1987 under accessing numbers 12 398 and 12399 respectively.

All the publications which are referred to in the present disclosure areincorporated herein by reference.

BIBLIOGRAPHIE

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1. A method of detecting HIV-2 retrovirus nucleic acid in a biologicalsample, said method comprising: a) contacting said sample with an HIV-2specific probe under stringent hybridization conditions, wherein saidprobe comprises an HIV-2 nucleic acid molecule, which hybridizes toHIV-2_(ROD) genomic DNA deposited as CNCM I-352 under stringenthybridization conditions; wherein the probe comprises an HIV-2 nucleicacid molecule obtained from nucleotides 1–380 of the U3/R region ofHIV-2, nucleotides 1–1566 of the gag gene of HIV-2, nucleotides1114–1524 of the gag gene, nucleotides 1–405 of the gag gene,nucleotides 406–1155 of the gag gene, or nucleotides 1–2673 of the envgene of HIV-2 or a fragment of said nucleic acid molecules; b) washingthe resulting hybrid; and c) detecting said hybrid.
 2. A method ofproducing an HIV-2 specific hybridization probe for HIV-2 retrovirusnucleic acid, said method comprising: a) providing a nucleic acidinsert, which hybridizes to HIV-2ROD genomic DNA deposited as CNCM I-352under stringent hybridization conditions, wherein the insert comprisesan HIV-2 nucleic acid molecule obtained from nucleotides 1–380 of theU3/R region of HIV-2, nucleotides 1–1566 of the gag gene of HIV-2,nucleotides 1114–1524 of the gag gene, nucleotides 1–405 of the gaggene, nucleotides 406–1155 of the gag gene, or nucleotides 1–2673 of theenv gene of HIV-2 or a fragment of said nucleic acid molecules; b)introducing the insert into a vector; c) introducing said vector into acompetent cellular host; d) culturing the cellular host; and e)recovering the DNA recombinants.
 3. The method of claim 1, wherein thehybridization occurs under conditions of 5×SSC, 5× Denhart, 50%formamide, at 42° C., and washing of the resulting hybrid occurs underconditions of 0.1×SSC, 0.1% SDS, at 65° C.
 4. The method of claim 2,wherein the hybridization occurs under conditions of 5×SSC, 5× Denhart,50% formamide, at 42° C. and washing of the resulting hybrid occursunder conditions of 0.1×SSC, 0.1% SDS, at 65° C.
 5. The method of anyone of claims 1 and 2, wherein said probe is obtained from the followingsequence: GTGGAAGGCG AGACTGAAAG CAAGAGGAAT ACCATTTAGT TAAAGGACAGGAACAGCTAT ACTTGGTCAG GGCAGGAAGT AACTAACAGA AACAGCTGAG ACTGCAGGGACTTTCCAGAA GGGGCTGTAA CCAAGGGAGG GACATGGGAG GAGCTGGTGG GGAACGCCTCATATTCTCTG TATAATATAC CCGCTGCTTG CATTGTACTT CAGTCGCTCT GCGGAGAGGCTGGCAGATTG AGCCCTGGAG GATCTCTCCA GCACTAGACG GATGAGCCTG GGTGCCCTGCTAGACTCTCA CCAGCACTTG GCCGGTGCTG GCAGACGGCC CCACGCTTGC CTGCTTAAAAACCTTCCTTA ATAAAGCTGC AGTAGAAGCA.


6. The method of any one of claims 1 and 2, wherein said probe encodesthe following amino acid sequence: Met Gly Ala Arg Asn Ser Val Leu ArgGly Lys Lys Ala Asp Glu Leu Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys LysLys Tyr Arg Leu Lys His Ile Val Trp Ala Ala Asn Lys Leu Asp Arg Phe GlyLeu Ala Glu Ser Leu Leu Glu Ser Lys Giu Gly Cys Gln Lys Ile Leu Thr ValLeu Asp Pro Met Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Phe Asn ThrVal Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys Asp Thr Glu GlyAla Lys Gln Ile Val Arg Arg His Leu Val Ala Glu Thr Gly Thr Ala Glu LysMet Pro Ser Thr Ser Arg Pro Thr Ala Pro Ser Ser Glu Lys Gly Gly Asn TyrPro Val Gln His Val Gly Gly Asn Tyr Thr His Ile Pro Leu Ser Pro Arg ThrLeu Asn Ala Trp Val Lys Leu Val Glu Glu Lys Lys Phe Gly Ala Glu Val ValPro Gly Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro Tyr Asp Ile Asn Gln MetLeu Asn Cys Val Gly Asp His Gln Ala Ala Met Gln Ile Ile Arg Glu Ile IleAsn Glu Glu Ala Ala Glu Trp Asp Val Gln His Pro Ile Pro Gly Pro Leu ProAla Gly Gln Leu Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser ThrVal Glu Glu Gln Ile Gln Trp Met Phe Arg Pro Gln Asn Pro Val Pro Val GlyAsn Ile Tyr Arg Arg Trp Ile Gln Ile Gly Leu Gln Lys Cys Val Arg Met TyrAsn Pro Thr Asn Ile Leu Asp Ile Lys Gln Gly Pro Lys Glu Pro Phe Gln SerTyr Val Asp Arg Phe Tyr Lys Ser Leu Arg Ala Glu Gln Thr Asp Pro Ala ValLys Asn Trp Met Thr Gln Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys LysLeu Val Leu Lys Gly Leu Gly Met Asn Pro Thr Leu Glu Glu Met Leu Thr AlaCys Gln Gly Val Gly Gly Pro Gly Gln Lys Ala Arg Leu Met Ala Glu Ala LeuLys Glu Val Ile Gly Pro Ala Pro Ile Pro Phe Ala Ala Ala Gln Gln Arg LysAla Phe Lys Cys Trp Asn Cys Gly Lys Glu Gly His Ser Ala Arg Gln Cys ArgAla Pro Arg Arg Gln Gly Cys Trp Lys Cys Gly Lys Pro Gly His Ile Met ThrAsn Cys Pro Asp Arg Gln Ala Gly Phe Leu Gly Leu Gly Pro Trp Gly Lys LysPro Arg Asn Phe Pro Val Ala Gln Val Pro Gln Gly Leu Thr Pro Thr Ala ProPro Val Asp Pro Ala Val Asp Leu Leu Glu Lys Tyr Met Gln Gln Gly Lys ArgGln Arg Glu Gln Arg Glu Arg Pro Tyr Lys Glu Val Thr Glu Asp Leu Leu HisLeu Glu Gln Gly Glu Thr Pro Tyr Arg Glu Pro Pro Thr Glu Asp Leu Leu HisLeu Asn Ser Leu Phe Gly Lys Asp Gln.


7. The method of any one of claims 1 and 2, wherein said probe encodesthe following amino acid sequence: Arg Lys Ala Phe Lys Cys Trp Asn CysGly Lys Glu Gly His Ser Ala Arg Gln Cys Arg Ala Pro Arg Arg Gln Gly CysTrp Lys Cys Gly Lys Pro Gly His Ile Met Thr Asn Cys Pro Asp Arg Gln AlaGly Phe Leu Gly Leu Gly Pro Trp Gly Lys Lys Pro Arg Asn Phe Pro Val AlaGln Val Pro Gln Gly Leu Thr Pro Thr Ala Pro Pro Val Asp Pro Ala Val AspLeu Leu Glu Lys Tyr Met Gln Gln Gly Lys Arg Gln Arg Glu Gln Arg Glu ArgPro Tyr Lys Glu Val Thr Glu Asp Leu Leu His Leu Glu Gln Gly Glu Thr ProTyr Arg Glu Pro Pro Thr Glu Asp Leu Leu His Leu Asn Ser Leu Phe Gly LysAsp Gln.


8. The method of any one of claims 1 and 2, wherein said probe encodesthe following amino acid sequence: Met Gly Ala Arg Asn Ser Val Leu ArgGly Lys Lys Ala Asp Glu Leu Glu Arg Ile Arg Leu Arg Pro Gly Gly Lys LysLys Tyr Arg Leu Lys His Ile Val Trp Ala Ala Asn Lys Leu Asp Arg Phe GlyLeu Ala Glu Ser Leu Leu Glu Ser Lys Glu Gly Cys Gln Lys Ile Leu Thr ValLeu Asp Pro Met Val Pro Thr Gly Ser Glu Asn Leu Lys Ser Leu Phe Asn ThrVal Cys Val Ile Trp Cys Ile His Ala Glu Glu Lys Val Lys Asp Thr Glu GlyAla Lys Gln Ile Val Arg Arg His Leu Val Ala Glu Thr Gly Thr Ala Glu LysMet Pro Ser Thr Ser Arg Pro Thr Ala Pro Ser Ser Glu Lys Gly Gly Asn Tyr.


9. The method of any one of claims 1 and 2, wherein said probe encodesthe following amino acid sequence: Pro Val Gln His Val Gly Gly Asn TyrThr His Ile Pro Leu Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Leu Val GluGlu Lys Lys Phe Gly Ala Glu Val Val Pro Gly Phe Gln Ala Leu Ser Glu GlyCys Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys Val Gly Asp His Gln AlaAla Met Gln Ile Ile Arg Glu Ile Ile Asn Glu Glu Ala Ala Glu Trp Asp ValGln His Pro Ile Pro Gly Pro Leu Pro Ala Gly Gln Leu Arg Glu Pro Arg GlySer Asp Ile Ala Gly Thr Thr Ser Thr Val Glu Glu Gln Ile Gln Trp Met PheArg Pro Gln Asn Pro Val Pro Val Gly Asn Ile Tyr Arg Arg Trp Ile Gln IleGly Leu Gln Lys Cys Val Arg Met Tyr Asn Pro Thr Asn Ile Leu Asp Ile LysGln Gly Pro Lys Glu Pro Phe Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser LeuArg Ala Glu Gln Thr Asp Pro Ala Val Lys Asn Trp Met Thr Gln Thr Leu LeuVal Gln Asn Ala Asn Pro Asp Cys Lys Leu Val Leu Lys Gly Leu Gly Met AsnPro Thr Leu Glu Glu Met Leu Thr Ala Cys Gln Gly Val Gly Gly Pro Gly GlnLys Ala Arg Leu Met Ala Glu Ala Leu Lys Glu Val Ile Gly Pro Ala Pro IlePro Phe Ala Ala Ala Gln Gln.


10. The method of any one of claims 1 and 2, wherein said probe encodesthe following amino acid sequence: Met Met Asn Gln Leu Leu Ile Ala IleLeu Leu Ala Ser Ala Cys Leu Val Tyr Cys Thr Gln Tyr Val Thr Val Phe TyrGly Val Pro Thr Trp Lys Asn Ala Thr Ile Pro Leu Phe Cys Ala Thr Arg AsnArg Asp Thr Trp Gly Thr Ile Gln Cys Leu Pro Asp Asn Asp Asp Tyr Gln GluIle Thr Leu Asn Val Thr Glu Ala Phe Asp Ala Trp Asn Asn Thr Val Thr GluGln Ala Ile Glu Asp Val Trp His Leu Phe Glu Thr Ser Ile Lys Pro Cys ValLys Leu Thr Pro Leu Cys Val Ala Met Lys Cys Ser Ser Thr Glu Ser Ser ThrGly Asn Asn Thr Thr Ser Lys Ser Thr Ser Thr Thr Thr Thr Thr Pro Thr AspGln Glu Gln Glu Ile Ser Glu Asp Thr Pro Cys Ala Arg Ala Asp Asn Cys SerGly Leu Gly Glu Glu Glu Thr Ile Asn Cys Gln Phe Asn Met Thr Gly Leu GluArg Asp Lys Lys Lys Gln Tyr Asn Glu Thr Trp Tyr Ser Lys Asp Val Val CysGlu Thr Asn Asn Ser Thr Asn Gln Thr Gln Cys Tyr Met Asn His Cys Asn ThrSer Val Ile Thr Glu Ser Cys Asp Lys His Tyr Trp Asp Ala Ile Arg Phe ArgTyr Cys Ala Pro Pro Gly Tyr Ala Leu Leu Arg Cys Asn Asp Thr Asn Tyr SerGly Phe Ala Pro Asn Cys Ser Lys Val Val Ala Ser Thr Cys Thr Arg Met MetGlu Thr Gln Thr Ser Thr Trp Phe Gly Phe Asn Gly Thr Arg Ala Glu Asn ArgThr Tyr Ile Tyr Trp His Gly Arg Asp Asn Arg Thr Ile Ile Ser Leu Asn LysTyr Tyr Asn Leu Ser Leu His Cys Lys Arg Pro Gly Asn Lys Thr Val Lys GlnIle Met Leu Met Ser Gly His Val Phe His Ser His Tyr Gln Pro Ile Asn LysArg Pro Arg Gln Ala Trp Cys Trp Phe Lys Gly Lys Trp Lys Asp Ala Met GlnGlu Val Lys Thr Leu Ala Lys His Pro Arg Tyr Arg Gly Thr Asn Asp Thr ArgAsn Ile Ser Phe Ala Ala Pro Gly Lys Gly Ser Asp Pro Glu Val Ala Tyr MetTrp Thr Asn Cys Arg Gly Glu Phe Leu Tyr Cys Asn Met Thr Trp Phe Leu AsnTrp Ile Glu Asn Lys Thr His Arg Asn Tyr Ala Pro Cys His Ile Lys Gln IleIle Asn Thr Trp His Lys Val Gly Arg Asn Val Tyr Leu Pro Pro Arg Glu GlyGlu Leu Ser Cys Asn Ser Thr Val Thr Ser Ile Ile Ala Asn Ile Asp Trp GlnAsn Asn Asn Gln Thr Asn Ile Thr Phe Ser Ala Glu Val Ala Glu Leu Tyr ArgLeu Glu Leu Gly Asp Tyr Lys Leu Val Glu Tie Thr Pro Ile Gly Phe Ala ProThr Lys Glu Lys Arg Tyr Ser Ser Ala His Gly Arg His Thr Arg Gly Val PheVal Leu Gly Phe Leu Gly Phe Leu Ala Thr Ala Gly Ser Ala Met Gly Ala ArgAla Ser Leu Thr Val Ser Ala Gln Ser Arg Thr Leu Leu Ala Gly Tie Val GlnGln Gln Gln Gln Leu Leu Asp Val Val Lys Arg Gln Gln Glu Leu Leu Arg LeuThr Val Trp Gly Thr Lys Asn Leu Gln Ala Arg Val Thr Ala Ile Glu Lys TyrLeu Gln Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg Gln Val CysHis Thr Thr Val Pro Trp Val Asn Asp Ser Leu Ala Pro Asp Trp Asp Asn MetThr Trp Gln Glu Trp Glu Lys Gln Val Arg Tyr Leu Glu Ala Asn Ile Ser LysSer Leu Glu Gln Ala Gln Ile Gln Gln Glu Lys Asn Met Tyr Glu Leu Gln LysLeu Asn Ser Trp Asp Ile Phe Gly Asn Trp Phe Asp Leu Thr Ser Trp Val LysTyr Ile Gln Tyr Gly Val Leu Ile Ile Val Ala Val Ile Ala Leu Arg Ile ValIle Tyr Val Val Gln Met Leu Ser Arg Leu Arg Lys Gly Tyr Arg Pro Val PheSer Ser Pro Pro Gly Tyr Ile Gln Gln Ile His Ile His Lys Asp Arg Gly GlnPro Ala Asn Glu Glu Thr Glu Glu Asp Gly Gly Ser Asn Gly Gly Asp Arg TyrTrp Pro Trp Pro Ile Ala Tyr Ile His Phe Leu Ile Arg Gln Leu Ile Arg LeuLeu Thr Arg Leu Tyr Ser Ile Cys Arg Asp Leu Leu Ser Arg Ser Phe Leu ThrLeu Gln Leu Ile Tyr Gln Asn Leu Arg Asp Trp Leu Arg Leu Arg Thr Ala PheLeu Gln Tyr Gly Cys Glu Trp Ile Gln Glu Ala Phe Gln Ala Ala Ala Arg AlaThr Arg Glu Thr Leu Ala Gly Ala Cys Arg Gly Leu Trp Arg Val Leu Glu ArgIle Gly Arg Gly Ile Leu Ala Val Pro Arg Arg Ile Arg Gln Gly Ala Glu IleAla Leu Leu *** Gly Thr Ala Val Ser Ala Gly Arg Leu Tyr Glu Tyr Ser MetGlu Gly Pro Ser Ser Arg Lys Gly Glu Lys Phe Val Gln Ala Thr Lys Tyr Gly,

wherein *** indicates a stop codon.
 11. The method of any one of claims1 or 2, wherein said probe comprises a cDNA or a fragment thereof.